CASH (CASPASE HOMOLOGUE) with death effector domain, modulators of the function of FAS

ABSTRACT

Proteins capable of modulating or mediating the function of MORT-1 are disclosed. Also disclosed are DNA sequences encoding these proteins, the recombinant production of these proteins as well as their use.

CROSS REFERENCE TO RELATED APPLICATION

The present application is a division of application Ser. No.10/849,901, filed May 21, 2004, which is a continuation of applicationSer. No. 09/380,546, filed Nov. 29, 1999, which is the national stageunder 35 U.S.C. 371 of PCT/IL98/00098, filed Feb. 26, 1998, which claimspriority from IL 120367/1997, filed Mar. 3, 1997 and IL 120759/1997,filed May 1, 1997. The entire contents of prior applications Ser. Nos.10/849,901, 09/380,546 and PCT/IL98/00098 are herein incorporated byreference.

FIELD OF THE INVENTION

The present invention is generally in the field of receptors belongingto the TNF/NGF superfamily of receptors and the control of theirbiological functions. The TNF/NGF superfamily of receptors includesreceptors such as the p55 and p75 tumor necrosis factor receptors(TNF-Rs, also called CD120a and CD120b, respectively, but hereinafterwill be called p55-R and p75-R) and the FAS ligand receptor (also calledFAS/APO1 or FAS-R or CD95. but hereinafter will be called FAS-R) andothers. More specifically, the present invention further concerns novelproteins which bind to other proteins which themselves bind to theprotein MORT-1 (or FADD), these also called MORT-1-binding proteins, or,which may also bind MORT-1 directly, and more specifically, it relatesto one such protein, herein designated G1, (which has now also beendesignated ‘CASH’ for ‘CASPASE HOMOLOG’, but which will be called ‘G1’herein throughout), which binds to the MORT-1-binding protein Mch4 (alsodesignated/called CASP-10), and possibly also to another MORT-1-bindingprotein called MACH (also designated/called CASP-8), and possibly alsodirectly to MORT-1 itself.

Accordingly, the present invention concerns, in general, new proteinswhich are capable of modulating or mediating the function of MORT-1directly or indirectly or of other proteins which bind to MORT-1directly or indirectly. In particular, the present invention concernsG1, its preparation and uses thereof, as well as the various novelisoforms of G1, their preparation and uses.

BACKGROUND OF THE RELATED ART

Tumor Necrosis Factor (TNF-α) and Lymphotoxin (TNF-β) (hereinafter, TNF,refers to both TNF-60 and TNF-β) are multifunctional pro-inflammatorycytokines formed mainly by mononuclear phagocytes, which have manyeffects on cells (Wallach, D. (1986) In: Interferon 7 (Ion Gresser,ed.), pp. 83-122, Academic Press, London; and Beutler and Cerami(1987)). Both TNF-a and TNF-β initiate their effects by binding tospecific cell surface receptors. Some of the effects are likely to bebeneficial to the organism: they may destroy, for example, tumor cellsor virus infected cells and augment antibacterial activities ofgranulocytes. In this way, TNF contributes to the defense of theorganism against tumors and infectious agents and contributes to therecovery from injury. Thus, TNF can be used as an anti-tumor agent inwhich application it binds to its receptors on the surface of tumorcells and thereby initiates the events leading to the death of the tumorcells. TNF can also be used as an anti-infectious agent.

However, both TNF-α and TNF-β also have deleterious effects. There isevidence that overproduction of TNF-α can play a major pathogenic rolein several diseases. For example, effects of TNF-α, primarily on thevasculature, are known to be a major cause for symptoms of septic shock(Tracey et al., 1986). In some diseases, TNF may cause excessive loss ofweight (cachexia) by suppressing activities of adipocytes and by causinganorexia, and TNF-α was thus called cachetin. It was also described as amediator of the damage to tissues in rheumatic diseases (Beutler andCerami, 1987) and as a major mediator of the damage observed ingraft-versus-host reactions (Piquet et al., 1987). In addition, TNF isknown to be involved in the process of inflammation and in many otherdiseases.

Two distinct, independently expressed, receptors, the p55 and p75TNF-Rs, which bind both TNF-α and TNF-β specifically, initiate and/ormediate the above noted biological effects of TNF. These two receptorshave structurally dissimilar intracellular domains suggesting that theysignal differently (See Hohmann et al., 1989; Engelmann et al., 1990;Brockhaus et al., 1990; Leotscher et al., 1990; Schall et al., 1990;Nophar et al., 1990; Smith et al., 1990; and Heller et al., 1990).However, the cellular mechanisms, for example, the various proteins andpossibly other factors, which are involved in the intracellularsignaling of the p55 an p75 TNF-Rs have yet to be elucidated. It is thisintracellular signaling, which occurs usually after the binding of theligand, i.e., TNF (α or β), to the receptor, that is responsible for thecommencement of the cascade of reactions that ultimately result in theobserved response of the cell to TNF.

As regards the above-mentioned cytocidal effect of TNF, in most cellsstudied so far, this effect is triggered mainly by the p55 TNF-R.Antibodies against the extracellular domain (ligand binding domain) ofthe p55 TNF-R can themselves trigger the cytocidal effect (see EP412486) which correlates with the effectivity of receptor cross-linkingby the antibodies, believed to be the first step in the generation ofthe intracellular signaling process. Further, mutational studies(Brakebusch et al., 1992; Tartaglia et al., 1993) have shown that thebiological function of the p55 TNF-R depends on the integrity of itsintracellular domain, and accordingly it has been suggested that theinitiation of intracellular signaling leading to the cytocidal effect ofTNF occurs as a consequence of the association of two or moreintracellular domains of the p55 TNF-R. Moreover, TNF (α and β) occursas a homotrimer, and as such, has been suggested to induce intracellularsignaling via the p55 TNF-R by way of its ability to bind to and tocross-link the receptor molecules, i.e., cause receptor aggregation.

Another member of the TNF/NGF superfamily of receptors is the FASreceptor (FAS-R) which has also been called the FAS antigen, acell-surface protein expressed in various tissues and sharing homologywith a number of cell-surface receptors including TNF-R and NGF-R. TheFAS-R mediates cell death in the form of apoptosis (Itoh et al., 1991),and appears to serve as a negative selector of autoreactive T cells,i.e., during maturation of T cells, FAS-R mediates the apoptopic deathof T cells recognizing self-antigens. It has also been found thatmutations in the FAS-R gene (lpr) cause a lymphoproliferation disorderin mice that resembles the human autoimmune disease systemic lupuserythematosus (SLE) (Watanabe-Fukunaga et al., 1992). The ligand for theFAS-R appears to be a cell-surface associated molecule carried by,amongst others, killer T cells (or cytotoxic T lymphocytes—CTLs), andhence when such CTLs contact cells carrying FAS-R, they are capable ofinducing apoptopic cell death of the FAS-R-carrying cells. Further, amonoclonal antibody has been prepared that is specific for FAS-R, thismonoclonal antibody being capable of inducing apoptopic cell death incells carrying FAS-R, including mouse cells transformed by cDNA encodinghuman FAS-R (Itoh et al., 1991).

While some of the cytotoxic effects of lymphocytes are mediated byinteraction of a lymphocyte-produced ligand with the widely occurringcell surface receptor FAS-R (CD95), which has the ability to triggercell death, it has also been found that various other normal cells,besides T lymphocytes, express the FAS-R on their surface and can bekilled by the triggering of this receptor. Uncontrolled induction ofsuch a killing process is suspected to contribute to tissue damage incertain diseases, for example, the destruction of liver cells in acutehepatitis. Accordingly, finding ways to restrain the cytotoxic activityof FAS-R may have therapeutic potential.

Conversely, since it has also been found that certain malignant cellsand HIV-infected cells carry the FAS-R on their surface, antibodiesagainst FAS-R, or the FAS-R ligand, may be used to trigger the FAS-Rmediated cytotoxic effects in these cells and thereby provide a meansfor combating such malignant cells or HIV-infected cells (see Itoh etal., 1991). Finding yet other ways for enhancing the cytotoxic activityof FAS-R may therefore also have therapeutic potential.

It has been a long felt need to provide a way for modulating thecellular response to TNF (α or β) and FAS-R ligand. For example, in thepathological situations mentioned above, where TNF or FAS-R ligand isoverexpressed, it is desirable to inhibit the TNF- or FAS-Rligand-induced cytocidal effects, while in other situations, e.g., woundhealing applications, it is desirable to enhance the TNF effect, or inthe case of FAS-R, in tumor cells or HIV-infected cells, it is desirableto enhance the FAS-R mediated effect.

A number of approaches have been made by the applicants (see forexample, European Application Nos. EP 186833, EP 308378, EP 398327 andEP 412486) to regulate the deleterious effects of TNF by inhibiting thebinding of TNF to its receptors using anti-TNF antibodies or by usingsoluble TNF receptors (being essentially the soluble extracellulardomains of the receptors) to compete with the binding of TNF to the cellsurface-bound TNF-Rs. Further, on the basis that TNF-binding to itsreceptors is required for the TNF-induced cellular effects, approachesby applicants (see for example EPO 568925) have been made to modulatethe TNF effect by modulating the activity of the TNF-Rs.

Briefly, EPO 568925 relates to a method of modulating signaltransduction and/or cleavage in TNF-Rs whereby peptides or othermolecules may interact either with the receptor itself or with effectorproteins interacting with the receptor, thus modulating the normalfunction of the TNF-Rs. In EPO 568925, there is described theconstruction and characterization of various mutant p55 TNF-Rs, havingmutations in the extracellular, transmembrane, and intracellular domainsof the p55 TNF-R. In this way, regions within the above domains of thep55 TNF-R were identified as being essential to the functioning of thereceptor, i.e., the binding of the ligand (TNF) and the subsequentsignal transduction and intracellular signaling which ultimately resultsin the observed TNF-effect on the cells. Further, there is alsodescribed a number of approaches to isolate and identify proteins,peptides or other factors which are capable of binding to the variousregions in the above domains of the TNF-R, which proteins, peptides andother factors may be involved in regulating or modulating the activityof the TNF-R. A number of approaches for isolating and cloning the DNAsequences encoding such proteins and peptides; for constructingexpression vectors for the production of these proteins and peptides;and for the preparation of antibodies or fragments thereof whichinteract with the TNF-R or with the above proteins and peptides thatbind various regions of the TNF-R, are also set forth in EPO 568925.However, EPO 568925 does not specify the actual proteins and peptideswhich bind to the intracellular domains of the TNF-Rs (e.g., p55 TNF-R),nor does it describe the yeast two-hybrid approach to isolate andidentify such proteins or peptides which bind to the intracellulardomains of TNF-Rs. Similarly, in EPO 568925 there is no disclosure ofproteins or peptides capable of binding the intracellular domain ofFAS-R.

Thus, when it is desired to inhibit the effect of TNF, or the FAS-Rligand, it would be desirable to decrease the amount or the activity ofTNF-Rs or FAS-R at the cell surface, while an increase in the amount orthe activity of TNF-Rs or FAS-R would be desired when an enhanced TNF orFAS-R ligand effect is sought. To this end the promoters of both the p55TNF-R and the p75 TNF-R have been sequenced, analyzed and a number ofkey sequence motifs have been found that are specific to varioustranscription regulating factors, and as such the expression of theseTNF-Rs can be controlled at their promoter level, i.e., inhibition oftranscription from the promoters for a decrease in the number ofreceptors, and an enhancement of transcription from the promoters for anincrease in the number of receptors (EP 606869 and WO 9531206).Corresponding studies concerning the control of FAS-R at the level ofthe promoter of the FAS-R gene have yet to be reported.

While it is known that the tumor necrosis factor (TNF) receptors, andthe structurally-related receptor FAS-R, trigger in cells, uponstimulation by leukocyte-produced ligands, destructive activities thatlead to their own demise, the mechanisms of this triggering are stilllittle understood. Mutational studies indicate that in FAS-R and the p55TNF receptor (p55-R) signaling for cytotoxicity involve distinct regionswithin their intracellular domains (Brakebusch et al., 1992; Tartagliaet al., 1993; Itoh and Nagata, 1993). These regions (the ‘death domains’or ‘death effector domains’ called ‘DED’) have sequence similarity. The‘death domains’ of both FAS-R and p55-R tend to self-associate. Theirself-association apparently promotes that receptor aggregation which isnecessary for initiation of signaling (see Song et al., 1994; Wallach etal., 1994; Boldin et al., 1995), and at high levels of receptorexpression can result in triggering of ligand-independent signaling(Boldin et al., 1995).

Some of the cytotoxic effects of lymphocytes are mediated by interactionof a lymphocyte-produced ligand with FAS-R (CD-95), a widely occurringcell surface receptor which has the ability to trigger cell death (seealso Nagata and Golstein, 1995); and that cell killing by mononuclearphagocytes involves a ligand-receptor couple, TNF and its receptor p55-R(CD120), that is structurally related to FAS-R and its ligand (see alsoVandenabeele et al., 1995). Like other receptor-induced effects, celldeath induction by the TNF receptors and FAS-R occurs via a series ofprotein-protein interactions, leading from ligand-receptor binding tothe eventual activation of enzymatic effector functions, which in thecase studies have elucidated non-enzymatic protein-protein interactionsthat intiate signaling for cell death:binding of trimeric TNF or theFAS-R ligand molecules to the receptors, the resulting interactions oftheir intracellular domains (Brakebusch et al., 1992; Tartaglia et al.,1993; Itoh and Nagata, 1993) augmented by a propensity of thedeath-domain motifs (or death effector domains, DED) to self-associate(Boldin et al., 1995a), and induced binding of two cytoplasmic proteins(which can also bind to each other) to the receptors' intracellulardomains—MORT-1 (or FADD) to FAS-R (Boldin et al., 1995b; Chinnaiyan etal., 1995; Kischkel et al., 1995) and TRADD to p55-R (Hsu et al., 1995;Hsu et al., 1996). Three proteins that bind to the intracellular domainof FAS-R and p55-R at the ‘death domain’ region involved in cell-deathinduction by the receptors through hetero-association of homologousregions and that independently are also capable of triggering cell deathwere identified by the yeast two-hybrid screening procedure. One ofthese is the protein, MORT-1 (Boldin et al. 1995b), also known as FADD(Chinnaiyan et al., 1995) that binds specifically to FAS-R. The secondone, TRADD (see also Hsu et al., 1995, 1996), binds to p55-R, and thethird, RIP (see also Stanger et al., 1995), binds to both FAS-R andp55-R. Besides their binding to FAS-R and p55-R, these proteins are alsocapable of binding to each other, which provides for a functional“cross-talk” between FAS-R and p55-R. These bindings occur through aconserved sequence motif, the ‘death domain module’ (also called ‘DED’for ‘Death Effector Domain’) common to the receptors and theirassociated proteins. Furthermore, although in the yeast two-hybrid testMORT-1 was shown to bind spontaneously to FAS-R, in mammalian cells,this binding takes place only after stimulation of the receptor,suggesting that MORT-1 participates in the initiating events of FAS-Rsignaling. MORT-1 does not contain any sequence motif characteristic ofenzymatic activity, and therefore, its ability to trigger cell deathseems not to involve an intrinsic activity of MORT-1 itself, but rather,activation of some other protein(s) that bind MORT-1 and act furtherdownstream in the signaling cascade. Cellular expression of MORT-1mutants lacking the N-terminal part of the molecule has been shown toblock cytotoxicity induction by FAS/APO1 (FAS-R) or p55-R (Hsu et al.,1996; Chinnaiyan et al., 1996), indicating that this N-terminal regiontransmits the signaling for the cytocidal effect of both receptorsthrough protein-protein interactions.

Recent studies have implicated a group of cytoplasmic thiol proteaseswhich are structurally related to the Caenorhabditis elegans proteaseCED3 and to the mammalian interleukin-1β converting enzyme (ICE) in theonset of various physiological cell death processes (reviewed in Kumar,1995 and Henkart, 1996). There have also been some indications thatprotease(s) of this family may take part in the cell-cytotoxicityinduced by FAS-R and TNF-Rs. Specific peptide inhibitors of theproteases and two virus-encoded proteins that block their function, thecowpox protein crmA and the Baculovirus p35 protein, were found toprovide protection to cells against this cell-cytotoxicity (Enari etal., 1995; Los et al., 1995; Tewari et al., 1995; Xue et al., 1995;Beidler et al., 1995). Rapid cleavage of certain specific cellularproteins, apparently mediated by protease(s) of the CED3/ICE family,could be demonstrated in cells shortly after stimulation of FAS-R orTNF-Rs.

It should be noted that these CED3/ICE proteases, also called caspases,are produced as inactive precursors and become activated by proteolyticprocessing upon death induction. These caspases are conserved cysteineproteases that cleave specific cellular proteins downstream of aspartateresidues thereby playing a critical role in all known programmed celldeath processes. In addition to their homologous C-terminal region fromwhich the mature proteases are derived, the precursor proteins containunique N-terminal regions. Interactions of these ‘prodomains’ withspecific regulatory molecules allow differential activation of thevarious caspases by different death-inducing signals (Boldin et al.,1996; Muzio et al., 1996; Duan and Dixit, 1997; Van Criekinge et al.,1996; Ahmad et al., 1997).

One such protease and various isoforms thereof (including inhibitoryones), designated MACH (also called CASP-8) which is a MORT-1 bindingprotein and which serves to modulate the activity of MORT-1 and hence ofFAS-R and p55-R, and which may also act independently of MORT-1, hasbeen recently isolated, cloned, characterized, and its possible usesalso described, as is set forth in detail and incorporated herein intheir entirety by reference, in co-owned, copending Israel PatentApplication Nos. IL 114615, 114986, 115319, 116588 and 117932, as wellas their corresponding PCT counterpart No. PCT/US96/10521, and in arecent publication of the present inventors (Boldin et al., 1996).Another such protease and various isoforms thereof (including inhibitoryones), designated Mch4 (also called CASP-10) has also recently beenisolated and characterized by the present inventors (unpublished) andothers (Fernandes-Alnemri et al., 1996; Srinivasula et al., 1996). ThisMch4 protein is also a MORT-1 binding protein which serves to modulatethe activity of MORT-1 and hence likely also of FAS-R and p55-R, andwhich may also act independently of MORT-1. Thus, details concerning allaspects, features, characteristics and uses of Mch4 are set forth in theabove noted publications, all of which are incorporated herein in theirentirety by reference.

It should also be noted that the caspases, MACH (CASP-8) and Mch4(CASP-10), which have similar prodomains (see Boldin et al., 1996; Muzioet al., 1996; Fernandes-Alnemri et al., 1996; Vincent and Dixit, 1997)interact through their prodomains with MORT-1, this interaction beingvia the ‘death domain motif’ or ‘death effector domain’, DED, present inthe N-terminal part of MORT-1 and present in duplicate in MACH (CASP-8)and Mch4 (CASP-10) (see Boldin et al., 1995b; Chinnalyan et al., 1995).

It should also be mentioned, in view of the above, that the variousproteins/enzymes/receptors involved in the intracellular signalingprocesses leading to cell death, have been given a variety of names. Inorder to precent confusion, the following is a list of the variousnames, including new names decided upon by a new convention, of each ofthese proteins, or parts thereof, and the names which are used hereinthroughout for convenience: Common or first name Other names NewConvention name Name used herein p55 TNF receptor p55-R CD120a p55-R p75TNF receptor p75-R CD120b p75-R FAS receptor FAS-R, FAS/APO1 CD95 FAS-RMORT-1 FADD — MORT-1 MACH FLICE1, Mch5 CASP-8 MACH Mch4 FLICE2 CASP-10Mch4 G1 — CASH G1 ‘death domain’ death domain motif, — deathdomain/death MORT motif, death domain motif/ effector domain (DED), MORTmodules MORT modules CED3/ICE proteases caspases CASP CED3/ICE proteases

SUMMARY OF THE INVENTION

It is an object of the invention to provide novel proteins, includingall isoforms, analogs, fragments or derivatives thereof, which arecapable of binding to MORT-1-binding proteins such as, for example, theabove noted Mch4 and MACH proteins and their isoforms, or which arecapable of binding to MORT-1 itself. As MORT-1 itself binds to theintracellular domain of the FAS-R, the novel proteins of the presentinvention by binding to the MORT-1-binding proteins and hence indirectlyto MORT-1, or by binding directly to the MORT-1 protein are thereforecapable of affecting the intracellular signaling process initiated bythe binding of the FAS ligand to its receptor, and as such the newproteins of the present invention are modulators of the FAS-R-mediatedeffect on cells. MORT-1 is also involved in the modulation of the TNFeffect on cells via its involvement in the modulation of p55-R and hencethe new proteins of the present invention are also conceived asmodulators of the TNF-effect, mediated by the p55-R, on cells. Likewise,by analogy to the above modulation of the FAS-R and p55-R mediatedeffect on cells, the proteins of the present invention may also bemediators or modulators of other cytotoxic mediators or inducers by wayof operating via common or related intracellular signaling pathways inwhich the proteins (e.g. G1 and its isoforms) of the invention areinvolved. These novel proteins of the present invention are designated“G1” proteins, and as noted above include the G1 protein (exemplifiedhereinbelow), all its isoforms, analogs, fragments or derivativesthereof.

Another object of the invention is to provide antagonists (e.g.,antibodies, peptides, organic compounds, or even some isoforms) to theabove novel G1 proteins, isoforms, analogs, fragments and derivativesthereof, which may be used to inhibit the signaling process, or, morespecifically, the cell-cytotoxicity, when desired.

A further object of the invention is to use the above novel G1 proteins,isoforms, analogs, fragments and derivatives thereof, to isolate andcharacterize additional proteins or factors, which may be involved inregulation of receptor activity, e.g., other proteases which cleave thenovel proteins to render then biologically active, and/or to isolate andidentify other receptors further upstream in the signaling process towhich these novel proteins, analogs, fragments and derivatives bind(e.g., other FAS-Rs or related receptors), and hence, in whose functionthey are also involved.

A still further object of the invention is to provide inhibitors whichcan be introduced into cells to bind or interact with the G1 protein andpossible G1 isoforms having protease activity (the G1 protein has aregion that is homologous to the proteolytic regions of Mch4 and MACH)and inhibit their intracellular activity which, at least for somepossible G1 isoforms, may be a proteolytic activity.

Moreover, it is an object of the present invention to use theabove-mentioned novel G1 proteins, isoforms and analogs, fragments andderivatives thereof as antigens for the preparation of polyclonal and/ormonoclonal antibodies thereto. The antibodies, in turn, may be used, forexample, for the purification of the new proteins from differentsources, such as cell extracts or transformed cell lines.

Furthermore, these antibodies may be used for diagnostic purposes, e.g.,for identifying disorders related to abnormal functioning of cellulareffects mediated by the FAS-R or other related receptors.

A further object of the invention is to provide pharmaceuticalcompositions comprising the above novel G1 proteins, isoforms, oranalogs, fragments or derivatives thereof, as well as pharmaceuticalcompositions comprising the above noted antibodies or other antagonists.

In accordance with the present invention, a novel protein, G1, which iscapable of binding to or, or interacting with, Mch4, which itself bindsto MORT-1, which binds to the intracellular domain of the FAS-R, wasisolated. G1 also may interact with another MORT-1-binding proteincalled MACH, and may also be capable of binding or interacting directlywith MORT-1. G1 probably functions as a modulator component of thecell-death pathway initiated by the binding of FAS ligand to FAS-R atthe cell surface, and this by virtue of the fact that it has aproteolytic region similar to the proteolytic regions of Mch4 and MACH,and hence G1 may also be an active intracellular protease. Further,depending on the transcription/translation processes in the expressionof G1, especially its proteolytic region, some isoforms of G1 may beexpressed without an active proteolytic region and as such may serve asantagonists of proteolytic activity mediated by, for example, Mch4 andMACH. Proteases of the CED3/ICE family have been implicated in theapoptopic processes triggered by FAS-R. MORT-1 (or FADD) binds to theintracellular domain of FAS-R upon activation of this receptor and thenovel G1 protein of the present invention binds to MORT-1-bindingproteins such as Mch4 and possibly also MACH or possibly directly toMORT-1. The G1 protein, cloned and characterized in accordance with thepresent invention, may exist in multiple isoforms, some of whichisoforms have a CED3/ICE homology region which has proteolytic activity(proteolytic domain), similar to those of Mch4 and some isoforms ofMACH, and which may cause the death of cells when expressed in thecells. Thus, activation of this novel CED3/ICE homolog (i.e., thevarious G1 isoforms having the proteolytic domain) by FAS-R (via director indirect MORT-1 interaction) appears to constitute an effectorcomponent of the FAS-R-mediated cell-death pathway.

Moreover, G1 also appears to function as an effector component of thecell-death pathway initiated by the binding of TNF to p55-R at the cellsurface, this by way of indirect mechanism of MORT-1 binding to TRADD, aprotein which binds to the intracellular domain of p55-R (Hsu et al.,1995), followed by or together with G1 binding to MORT-1-bindingproteins such as, for example, Mch4 or MACH, or binding to MORT-1directly, with the activation of G1 into an active protease involved ineffecting cell death.

It should also be noted that while G1 displays at least some of thesequence features critical of the function of the CED3/ICE proteases, itdoes, however, have some distinctive sequence features of its own whichmay endow it with a unique and possibly tissue/cell specific mode ofaction.

Thus, in accordance with the present invention, a new protein designatedG1 is provided. This G1 protein was isolated and cloned by thetwo-hybrid screening assay and characterized as a molecule which bindsMch4. Mch4, as noted above, is a MORT-1-binding protein which is capableof effecting cell death, although however, it should also be noted thatsome isoforms of Mch4 have the opposite effect, namely, they inhibit thekilling of cells. Further, the sequencing of G1 has so far revealed thatit has in its N-terminal region two so-called ‘MORT MODULES’ (MM) whichare also found in the MORT-1-binding proteins MACH and Mch4. These MORTMODULES in G1 appear to account for its ability to bind to Mch4, and mayalso be the basis for its possible binding to MORT-1 directly and forits binding to MACH or to specific MACH isoforms (specific splicevariants of MACH). In the G1 sequence downstream of the N-terminusregion containing the MORT MODULES there also appears to be a longregion displaying similarity to proteolytic region of MACH and Mch4.Moreover, from an initial analysis of the possible location of the G1sequence in the human chromosomes, it appears that G1 is located onchromosome No. 2 very close to the positions of Mch4 and MACH which isalso indicative of a relationship between G1, MACH and Mch4.

More specifically, at least three possible isoforms of G1 have beenfound in accordance with the present invention (see Example 1 below).Two of these have been isolated and cloned and appear to represent twosplice variants of a novel protein, designated G1 (or CASH). These twoisoforms are called G1α (or CASHα) for the larger isoform and G1β (orCASHβ) for the shorter isoform (although there appears to be more thanone short isoform, hence G1β is also designated G1β1, and the othershort isoform is designated G1β2—see Example 1 below). These G1α and βisoforms each contain two N-terminal death domain motifs/MORT MODULESand can bind to each other via these death domain motifs, and can alsobind to MORT1, MACH and Mch4 via these death domain motifs. The longerG1α isoform has a unique C-terminal portion (in comparison to theshorter G1β isoform) this unique C-terminal portion having sequencehomology to the caspase protease activity region. With respect tobiological activity, the shorter G1β isoform inhibits celldeath/cytotoxicity mediated by p55-R and FAS-R, while, in contrast, thelonger G1α isoform has a cytotoxic effect on at least some types ofcells (e.g. 293 cells) which cytotoxicity involved its protease-homologyregion. However, it should also be noted that the longer G1α isoform isalso capable of inhibiting cytotoxicity mediated by FAS-R and p55-R inother types of cells (e.g. HeLa cells). These results indicate that G1(namely, its various isoforms) acts as an attenuator/inhibitor and/or aninitiator/enhancer of p55-R- and FAS-R-mediated signaling for celldeath.

It should also be noted that for the sake of clarity the variousisoforms of G1, for example G1α and G1β, will often be referred toherein as simply ‘G1’, but it is to be understood that in these casesall the isoforms of G1 are to be included in the meaning of ‘G1’ so thatthis could mean both inducers/enhancers of cell cytotoxicity as well asinhibitors/attenuators of cell cytotoxicity. When a specific G1 isoformis intended, then it will be named specifically, e.g. G1α or G1β, as thecase may be). As such ‘G1’ when used collectively to refer to thevarious isoforms will also often be referred to herein as a ‘modulator’,this meaning that it can be inhibitory or augmentory to the biologicalactivity in question.

In view of the above-mentioned, it therefore arises, as noted above andas set forth hereinbelow, that G1 is apparently a modulator of MORT-1activity and hence a modulator of the cellular effects mediated by theFAS-R and also the p55-R as well as possibly other receptors of theTNF/NGF receptor family and others as well which may share commonintracellular signaling components and mechanisms.

Thus, as G1 apparently has a protease-like region (at least the longisoform) it may be responsible directly for cell cytotoxicity andinflammation caused or induced by various stimuli including thosetransmitted via receptors of the TNF/NGF receptor family and possiblyothers as well.

G1 may also serve as an inhibitor of cell cytotoxicity and inflammationby virtue of its being present as part of a complex of other proteinsand as such may effect the cytotoxicity or inflammatory effects of theseother proteins (e.g. MACH and Mch4 or even MORT-1), ultimately resultingin an inhibition of their cytotoxic activity or their activity ininflammation.

G1 may yet also serve as an enhancer or augmentor of cell cytotoxicityand inflammation and this by augmenting the activity of other proteins(e.g. Mch4 and MACH or even MORT-1) by binding to them and recruitingthem to bind MORT-1 or to act independently of MORT-1, in either casethe recruitment serving to augment the cytotoxic activity of the variousproteins or to augment their inflammatory effects.

Likewise, in an analogous fashion G1 may also serve as an inhibitor oran augmentor of other intracellular mediators or modulators havingpathways in which G1 is actively involved.

MORT-1 (for ‘Mediator of Receptor Toxicity’, Boldin et al., 1995b), iscapable of binding to the intracellular domain of the FAS-R. ThisFAS-binding protein appears to act as a mediator or modulator of theFAS-R ligand effect oncells by way of mediating or modulating theintracellular signaling process which usually occurs following thebinding of the FAS-R ligand at the cell surface. In addition to itsFAS-binding specificity, MORT-1 was shown to have other characteristics(see Reference Example 1), for example, it has a region homologous tothe “death domain” (DD) regions of the p55-TNF-R and FAS-R (p55-DD andFAS-DD), and thereby is also capable of self-association. MORT-1 is alsocapable of activating cell cytotoxicity on its own, an activity possiblyrelated to its self-association capability. It has also been found thatco-expression of the region in MORT-1 that contains the “death domain”homology sequence (MORT-DD, present in the C-terminal part of MORT-1)strongly interferes with FAS-induced cell death, as would be expectedfrom its ability to bind to the “death domain” of the FAS-IC. Further,in the same experimental conditions, it was found that co-expression ofthe part of MORT-1 that does not contain the MORT-DD region (theN-terminal part of MORT-1, amino acids 1-117, “MORT-1 head”) resulted inno interference of the FAS-induced cell death and, if at all, a somewhatenhanced FAS-induced cell cytotoxicity.

Accordingly, it is likely that MORT-1 also binds to other proteinsinvolved in the intracellular signaling process. These MORT-1-bindingproteins may therefore also act as indirect mediators or modulators ofthe FAS-R ligand effect on cells by way of mediating or modulating theactivity of MORT-1; or these MORT-1-binding proteins may act directly asmediators or modulators of the MORT-1-associated intracellular signalingprocess by way of mediating or modulating the activity of MORT-1, which,as noted above, has an apparently independent ability to activate cellcytotoxicity. These MORT-1-binding proteins may also be used in any ofthe standard screening procedures to isolate, identify and characterizeadditional proteins, peptides, factors, antibodies, etc., which may beinvolved in the MORT-1-associated or FAS-R-associated signaling processor may be elements of other intracellular signaling processes. SuchMORT-1-binding proteins have been isolated and have been described asnoted above in the co-owned co-pending Israel application Nos. IL114,615, 114,986, 115,319, 116,588, 117,932 and their corresponding PCTapplication PCT/US96/10521 (as regards MACH and its isoforms), and byothers such as Fernandes-Alnemr et al. (1996) and Srinivasula et al.(1996) (as regards Mch4 and other such ‘Mch’ proteins). One of theseMORT-1-binding proteins, and above noted MACH, was initially cloned,sequenced, and partially characterized as having the followingproperties : The MACH cDNA encodes the ORF-B open-reading frame; MACHbinds to MORT-1 in a very strong and specific manner; the MACH bindingsite in MORT-1 occurs upstream of the MORT-1 “death domain” motif; theORF-B region of MACH is the MORT-1-interacting part thereof; and MACH iscapable of self-association and of inducing cell cytotoxicity on itsown. Further, later analysis as set forth in the above co-owned,co-pending patent applications as well as Boldin et al. (1996) showedthat MACH actually exists in a number of isoforms. Moreover, the MACHORF-B noted above is in fact one of the MACH isoforms designated asMACHβ1. In the above publications concerning Mch4 it was also shown thatthis protein also binds MORT-1 (or FADD) and is directly involved incell-cytotoxicity with MORT-1 or independent thereof and this by virtueof its proteolytic activity.

Accordingly, the present invention provides a DNA sequence encoding a G1protein, analogs or fragments thereof, capable of binding to orinteracting directly or indirectly with MORT-1 and/or any of theMORT-1-binding proteins, such as, for example, Mch4 or MACH, said G1protein, analogs or fragments thereof being capable of mediating theintracellular effect mediated by the FAS-R or p55-TNF-R, said G1protein, analogs or fragments thereof, also being capable of modulatingor mediating the intracellular effect of other intracellular proteins towhich it is capable of binding directly or indirectly.

In particular, the present invention provides a DNA sequence selectedfrom the group consisting of:

(a) a cDNA sequence derived from the coding region of a native G1protein;

(b) DNA sequences capable of hybridization to a sequence of (a) undermoderately stringent conditions and which encode a biologically activeG1 protein; and

(c) DNA sequences which are degenerate as a result of the genetic codeto the DNA sequences defined in (a) and (b) and which encode abiologically active G1 protein.

Another specific embodiment of the above DNA sequence of the inventionis a DNA sequence comprising at least part of the sequence encoding atleast one isoform of the G1 protein. Another embodiment of the above DNAsequence is the sequence encoding the G1 protein as depicted in FIG. 1,(the G1α isoform). Another such embodiment is a second G1 isoformdepicted in FIG. 2 (the G1β isoform).

The present invention provides G1 proteins, and analogs, fragments orderivatives thereof encoded by any of the above sequences of theinvention, said proteins, analogs, fragments and derivatives beingcapable of binding to or interacting directly or indirectly with MORT-1and/or any of the MORT-1-binding proteins such as, for example, Mch4 orMACH, and mediating the intracellular effect mediated by the FAS-R orp55 TNF-R, or any other cytotoxic mediator of inducer to which said G1proteins, analogs, fragments or derivatives are capable of bindingdirectly or indirectly.

A specific embodiment of the invention is the G1 protein, analogs,fragments and derivatives thereof. Another embodiment is any isoform ofthe G1 protein, analogs, fragments and derivatives thereof.

Also provided by the present invention are vectors encoding the above G1protein, and analogs, fragments or derivatives of the invention, whichcontain the above DNA sequence of the invention, these vectors beingcapable of being expressed in suitable eukaryotic or prokaryotic hostcells; transformed eukaryotic or prokaryotic host cells containing suchvectors; and a method for producing the G1 protein, or analogs,fragments or derivatives of the invention by growing such transformedhost cells under conditions suitable for the expression of said protein,analogs, fragments or derivatives, effecting post-translationalmodifications of said protein as necessary for obtaining said proteinand extracting said expressed protein, analogs, fragments or derivativesfrom the culture medium of said transformed cells or from cell extractsof said transformed cells. The above definitions are intended to includeall isoforms of the G1 protein.

In another aspect, the present invention also provides antibodies oractive derivatives or fragments thereof specific the G1 protein, andanalogs, fragments and derivatives thereof, of the invention.

By yet another aspect of the invention, there are provided various usesof the above DNA sequences or the proteins which they encode, accordingto the invention, which uses include, in general, amongst others:

(A) A method for the modulation of cell death or inflammatory processes,comprising treating said cells with one or more G1 proteins, analogs,fragments or derivatives of the invention as noted above, wherein saidtreating of said cells comprises introducing into said cells said one ormore proteins, analogs, fragments or derivatives in a form suitable forintracellular introduction thereof, or introducing into said cells anucleotide sequence encoding said one or more proteins, analogs,fragments, or derivatives in the form of a suitable vector carrying saidsequence, said vector capable of effecting the insertion of saidsequence into said cells in a way that said sequence is expressed insaid cells; and

(B) A method for the modulation of cell death or inflammatory processes,comprising treating said cells with one or more inhibitors of one ormore proteins/enzymes mediating said cell death or inflammatoryprocesses, said inhibitors being selected from the group consisting of :(i) one or more G1 proteins, analogs, fragments or derivatives of theinvention, capable of inhibiting said cell death or inflammatoryprocesses; and (ii) inhibitors of one or more G1 proteins of theinvention when said one or more G1 proteins augments/enhances ormediates said cell death or inflammatory processes.

More particularly, the above methods of the present invention includethe following specific embodiments:

(i) A method for the modulation of the FAS-R ligand or TNF effect oncells carrying a FAS-R or p55-R, comprising treating said cells with oneor more G1 proteins, analogs, fragments or derivatives of the invention,capable of binding to MORT-1, directly or indirectly or capable ofbinding to MORT-1 binding proteins such as Mch4 or MACH, which MORT-1,in turn, directly binds to the intracellular domain of FAS-R, or capableof binding directly or indirectly to MORT-1 or to MORT-1-bindingproteins as noted above, which MORT-1, in turn, binds to TRADD whichbinds to the intracellular domain of p55-R, and thereby being capable ofmodulating/mediating the activity of said FAS-R or p55 TNF-R, whereinsaid treating of said cells comprises introducing into said cells saidone or more proteins, analogs, fragments or derivatives in a formsuitable for intracellular introduction thereof, or introducing intosaid cells a DNA sequence encoding said one or more proteins, analogs,fragments or derivatives in the form of a suitable vector carrying saidsequence, said vector being capable of effecting the insertion of saidsequence into said cells in a way that said sequence is expressed insaid cells.

(ii) A method for the modulation of the FAS-R ligand or TNF effect oncells according to (i) above, wherein said treating of cells comprisesintroducing into said cells said G1 protein, or analogs, fragments orderivatives thereof, in a form suitable for intracellular introduction,or introducing into said cells a DNA sequence encoding said G1 protein,or analogs, fragments or derivatives in the form of a suitable vectorcarrying said sequence, said vector being capable of effecting theinsertion of said sequence into said cells in a way that said sequenceis expressed in said cells.

(iii) A method as in (ii) above wherein said treating of said cells isby transfection of said cells with a recombinant animal virus vectorcomprising the steps of:

-   -   (a) constructing a recombinant animal virus vector carrying a        sequence encoding a viral surface protein (ligand) that is        capable of binding to a specific cell surface receptor on the        surface of a FAS-R- or p55-R-carrying cell and a second sequence        encoding a protein selected from G1 protein, and analogs,        fragments and derivatives thereof, that when expressed in said        cells ia capable of modulating/mediating the activity of said        FAS-R or p55-R; and    -   (b) infecting said cells with said vector of (a).

(iv) A method for modulating the FAS-R ligand of TNF effect on cellscarrying a FAS-R or a p55-R comprising treating said cells withantibodies or active fragments or derivatives thereof, according to theinvention, said treating being by application of a suitable compositioncontaining said antibodies, active fragments or derivatives thereof tosaid cells, wherein when at least part of the G1 protein is exposed onthe extracellular surface, said composition is formulated forextracellular application, and when said G1 proteins are entirelyintracellular, said composition is formulated for intracellularapplication.

(v) A method for modulating the FAS-R ligand or TNF effect on cellscarrying a FAS-R or p55-R comprising treating said cells with anoligonucleotide sequence encoding an antisense sequence of at least partof the G1 protein sequence of the invention, said oligonucleotidesequence being capable of blocking the expression of the G1 protein.

(vi) A method as in (ii) above for treating tumor cells or HIV-infectedcells or other diseased cells, comprising:

-   -   (a) constructing a recombinant animal virus vector carrying a        sequence encoding a viral surface protein capable of binding to        a specific tumor cell surface receptor or HIV-infected cell        surface receptor or receptor carried by other diseased cells and        a sequence encoding a protein selected from G1 protein, analogs,        fragments and derivatives of the invention, that when expressed        in said tumor, HIV-infected, or other diseased cell is capable        of killing said cell; and    -   (b) infecting said tumor or HIV-infected cells or other diseased        cells with said vector of (a).

(vii) A method for modulating the FAS-R ligand or TNF effect on cellscomprising applying the ribozyme procedure in which a vector encoding aribozyme sequence capable of interacting with a cellular mRNA sequenceencoding a G1 protein according to the invention, is introduced intosaid cells in a form that permits expression of said ribozyme sequencein said cells, and wherein when said ribozyme sequence is expressed insaid cells it interacts with said cellular mRNA sequence and cleavessaid mRNA sequence resulting in the inhibition of expression of said G1protein in said cells.

(viii) A method selected from the method according to the invention,wherein said G1 protein encoding sequence comprises at least one of theG1 isoforms, analogs, fragments and derivatives of any thereof accordingto the invention which are capable of binding directly or indirectly toMORT-1 or MORT-1-binding proteins such as, for example, Mch4 and MACH,which MORT-1, in turn, binds specifically to FAS-IC, or which arecapable of binding directly or indirectly to MORT-1 or the aboveMORT-1-binding proteins, which MORT-1, in turn, binds to TRADD and whichin turn binds to the p55-IC.

(ix) A method for isolating and identifying proteins, according to theinvention capable of binding directly or indirectly to the MORT-1protein or the MORT-1-binding proteins, comprising applying the yeasttwo-hybrid procedure in which a sequence encoding said MORT-1 protein orMORT-1-binding proteins is carried by one hybrid vector and sequencefrom a cDNA or genomic DNA library is carried by the second hybridvector, the vectors then being used to transform yeast host cells andthe positive transformed cells being isolated, followed by extraction ofthe said second hybrid vector to obtain a sequence encoding a proteinwhich binds to said MORT-1 protein or said MORT-1 -binding proteins.

(x) A method according to any of the (i)-(ix) above wherein said G1protein is any one of the isoforms of G1, analogs, fragments andderivatives of any thereof.

(xi) A method according to any of the above (i)-(x) wherein the G1protein or any of its isoforms, analogs, fragments or derivatives isinvolved in the modulation of the cellular effect mediated or modulatedby any other cytotoxic mediator or inducer to which said G1 protein,isoform, analog, fragment or derivative is capable of binding directlyor indirectly.

(xii) A method for screening other substances such as, for example,peptides, organic compounds, antibodies, etc. to obtain specific drugswhich are capable of inhibiting the activity of G1, e.g. inhibiting G1αprotease activity thereby inhibiting cell cytotoxicity, or inhibitingG1β activity thereby enhancing cell cytotoxicity.

Embodiments of the above screening method of (xii) include:

-   -   (1) A method for screening of a ligand capable of binding to a        G1 protein of the invention as noted above, comprising        contacting an affinity chromatography matrix to which said        protein is attached with a cell extract whereby the ligand is        bound to said matrix, and eluting, isolating and analyzing said        ligand.    -   (2) A method for screening of a DNA sequence coding for a ligand        capable of binding to a G1 protein of the invention, comprising        applying the yeast two-hybrid procedure in which a sequence        encoding said protein is carried by one hybrid vector and        sequences from a cDNA or genomic DNA library are carried by the        second hybrid vector, transforming yeast host cells with said        vectors, isolating the positively transformed cells, and        extracting said second hybrid vector to obtain a sequence        encoding said ligand.    -   (3) A method for identifying and producing a ligand capable of        modulating the cellular activity modulated/mediated by MORT-1 or        MORT-1-binding proteins comprising:        -   a) screening for a ligand capable of binding to a            polypeptide comprising at least a portion of MORT-1 or            MORT-1-binding proteins selected from MACH proteins, Mch4            proteins and other MORT-1-binding proteins;        -   b) identifying and characterizing a ligand, other than            MORT-1 or said MORT-1-binding proteins or portions of a            receptor of the TNF/NGF receptor family, found by said            screening step to be capable of said binding; and        -   c) producing said ligand in substantially isolated and            purified form.    -   (4) A method for identifying and producing a ligand capable of        modulating the cellular activity modulated or mediated by a G1        protein of the invention, comprising:        -   a) screening for a ligand capable of binding to a            polypeptide comprising at least a portion of the G1α            sequence depicted in FIG. 1 or at least a portion of the G1β            sequence depicted in FIG. 2;        -   b) identifying and characterizing a ligand, other than            MORT-1 or MORT-1-binding proteins or portions of a receptor            of the TNF/NGF receptor family, found by said screening step            to be capable of said binding; and        -   c) producing said ligand in substantially isolated and            purified form.    -   (5) A method for identifying and producing a ligand capable of        modulating the cellular activity modulated/mediated by G1        comprising:        -   a) screening for a ligand capable of binding to at least a            portion of the G1α sequence depicted in FIG. 1 or the G1β            sequence depicted in FIG. 2;        -   b) identifying and characterizing a ligand, other than            MORT-1 or MORT-1-binding proteins or portions of a receptor            of the TNF/NGF receptor family, found by said screening step            to be capable of said binding; and        -   c) producing said ligand in substantially isolated and            purified form.    -   (6) A method for identifying and producing a molecule capable of        directly or indirectly modulating the cellular activity        modulated/mediated by G1, comprising:        -   a) screening for a molecule capable of modulating activities            modulated/mediated by G1        -   b) identifying and characterizing said molecule; and        -   c) producing said molecule in substantially isolated and            purified form.    -   (7) A method for identifying and producing a molecule capable of        directly or indirectly modulating the cellular activity        modulated/mediated by a G1 protein of the invention, comprising:        -   a) screening for a molecule capable of modulating activities            modulated/mediated by said G1 protein;        -   b) identifying and characterizing said molecule; and        -   c) producing said molecule in substantially isolated and            purified form.

The present invention also provides a pharmaceutical composition for themodulation of the FAS-R ligand- or TNF-effect on cells or the effect ofany other cytotoxic mediator or inducer on cells as noted above,comprising, as active ingredient any one of the following:

(i) a G1 protein according to the invention, and biologically activefragments, analogs, derivatives of mixtures thereof;

(ii) a recombinant animal virus vector encoding a protein capable ofbinding a cell surface receptor and encoding a G1 protein orbiologically active fragments or analogs, according to the invention;

(iii) an oligonucleotide sequence encoding an anti-sense sequence of theG1 protein sequence according to the invention, wherein saidoligonucleotide may be the second sequence of the recombinant animalvirus vector of (ii) above.

The present invention also provides:

I. a method for the modulation of the MORT-1-induced effect orMORT-1-binding protein-induced effect, or the effect of any othercytotoxic mediator or inducer, on cells comprising treating said cellsin accordance with a method of any one of (i)-(x) above, with G1proteins, analogs, fragments or derivatives thereof or with sequencesencoding G1 proteins, analogs or fragments thereof, said treatmentresulting in the enhancement or inhibition of said MORT-1-mediatedeffect, and thereby also of the FAS-R or p55-R-mediated effect, or ofsaid other cytotoxic mediator or inducer.

II. a method as above wherein said G1 protein, analog, fragment orderivative thereof is that part of the G1 protein which is specificallyinvolved in binding to MORT-1 or MORT-1-binding proteins, or said othercytotoxic mediator or inducer, or said G1 protein sequence encodes thatpart of G1 protein which is specifically involved in binding to MORT-1or the MORT-1-binding proteins, or said other cytotoxic mediator orinducer.

III. a method as above wherein said G1 protein is any one of the G1isoforms, said isoforms capable of enhancing the MORT-1-associatedeffect, or other cytotoxic mediator or inducer associated effect oncells and thereby also of enhancing the FAS-R- or p55-R-associatedeffect on cells, or the other cytotoxic mediator or inducer effect oncells.

IV. a method as above wherein said G1 protein is any one of the G1isoforms, said isoforms capable of inhibiting the MORT-1-associatedeffect, or other cytotoxic mediator or inducer associated effect oncells and thereby also of inhibiting the FAS-R- or p55-R-associatedeffect on cells, or the other cytotoxic mediator or inducer effect oncells.

As arises from all the above-mentioned, as well as from the detaileddescription hereinbelow, G1 may also be used in a MORT-1 independentfashion to treat cells or tissues. Isolation of the G1 proteins, theiridentification and characterization may be carried out by any of thestandard screening techniques used for isolating and identifyingproteins, for example, the yeast two-hybrid method, affinitychromatography methods, and any of the other well-known standardprocedures used for this purpose.

Furthermore, some isoforms of G1 may have only a protease-like region(with homology to the above mentioned protease regions of other knownproteases) but which has no actual protease activity, with the resultthat such isoforms may serve primarily an inhibitory role as notedabove.

Moreover, as G1 or any of its isoforms may be involved in modulatingMORT-1-independent intracellular pathways, G1 or any of its isoforms maybe involved in the modulation of the signaling of any otherintracellular pathways or mechanisms.

Other aspects and embodiments of the present invention are also providedas arising from the following detailed description of the invention.

It should be noted that, where used throughout, the following terms:“Modulation of the FAS-ligand or TNF effect on cells”; and “Modulationof the MORT-1 or MORT-1-binding protein effect on cells” are understoodto encompass in vitro as well as in vivo treatment and, in addition,also to emcompass inhibition or enhancement/augmentation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A depicts schematically the preliminary nucleotide sequence (SEQID NO:1) of one isoform of the G1 protein as detailed in Example 1.

FIG. 1B depicts schematically the deduced amino acid sequence (SEQ IDNO:2) of the isoform depicted in FIG. 1A.

FIG. 2 depicts schematically the preliminary nucleotide (SEQ ID NO:3)and deduced amino acid sequence (SEQ ID NO:4) of the second isoform ofthe G1 protein as detailed in Example 1.

FIG. 3 depicts schematically a comparison of the amino acid sequences ofthe human (hCASHα, hCASHβ) G1 (or CASH) and mouse (mCASHα) splicevariants, and conserved motifs found in these proteins. In FIG. 3 thereis shown a collinear amino acid sequence alignment of mouse G1α (mCASHα)(SEQ ID NO:5), human G1α (hCASHα) (SEQ ID NO:2) and G1β (hCASHβ) (SEQ IDNO:4), CASP-8 (MACH/FLICE1/Mch5) (SEQ ID NO:6), CASP-10 (Mch4/FLICE2)(SEQ ID NO:7), CASP-3 (CPP32/Apopain/Yama) (SEQ ID NO:8) and CASP-1(ICE) (SEQ ID NO:9). CASP-1 and CASP-3 are shown without their prodomainregions. Amino acid residues are numbered to the right of each sequence.Dotted lines indicate gaps in the sequence to allow optimal alignment.The ‘death domain’ modules (DED) are shaded. Amino acids that areidentical in more than three of the proteins shown are boxed. Within theregion of protease homology, amino acids aligned with CASP-1 residuesthat were implicated in catalytic activity by X-ray crystallography aredenoted as follows: The residues putatively involved in catalysis,corresponding to His237 and Cys285 in CASP-1, are darkly shaded andmarked by closed circles below the alignment. The residues constitutingthe binding pocket for the carboxylate side chain of the P1 Asp,corresponding to Arg179, Gln 238, Arg341 and Ser347 in CASP-1, are lessheavily shaded and marked by open circles. Known and suggested Asp-Xcleavage sites and the potential site of cleavage found at a similarlocation in G1 (CASH) are shaded. Horizontal arrows indicate the N- andC-terminal ends of the small and large subunits of the CASP-1. TheC-termini of the proteins are denoted by asterisks.

FIG. 4 (A, B) shows reproductions of autoradiograms of Northern blotsdepicting the identification of G1 transcripts in various human tissues(heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,spleen, thymus, prostate, testis, ovary, small intestine, colon andperipheral blood lymphocytes—PBL). The Northern blot analysis wasperformed as follows: A radiolabeled mRNA probe corresponding to the‘death domain’ (DED) module region of G1 (from nucleotide nos. 482-1070in G1β (see FIG. 2), a region common to both G1 (CASH) splice variantscloned) was prepared using the T7 RNA polymerase (Promega) and used foranalysis of human multiple tissue blots (Clontech), containing poly(A)+RNA (2 μg per lane) of various human tissues.

FIG. 5 is a schematic presentation of the results showing theinteraction of G1α (CASHα) and G1β (CASHβ) with other ‘death domain’(DED)-containing proteins within transfected yeast (e.g. MORT1/FADD,MACHα1 (CASP-8α1), MACHβ1 (CASP-8β1), MACHβ4 (CASP-8β4), Mch4 (CASP-10),G1α (CASHα), G1β (CASHβ), p55-R (p55ic), RIP, TRADD and Lamin (negativecontrol)). The binding properties of G1β (CASHβ), as well as G1α (CASHα)were assessed in the yeast SFY526 reporter strain (Clontech), using thepGBT9-GAL4 DNA-binding domain and the pGAD1318 and pGADGH-GAL4activation-domain vectors. Quantification of the binding in yeast by theβ-galactosidase expression filter assay was performed as noted in theReference Examples 1-3. Results are expressed as the time required fordevelopment of strong color. In all cases tested, identical results wereobtained when placing the tested inserts in; the DNA-binding domain andactivation-domain constructs in both combinations. None of the examinedinserts interacted with several control proteins, including theintracellular domains of human p55-R (CD120a), p75-R (CD120b), CD40,lamin, and empty Gal4 vectors.

FIG. 6 (A-D) are presentations of the results showing the effects of G1α(CASHα), G1β (CASHβ) and G1 a (CASHα) mutants on cell viability and celldeath induction. Quantification of cell death induced in HeLa-Fas cells(results depicted schematically as bar-graphs in FIG. 6A) and in 293-Tcells (results depicted schematically as bar-graphs in FIGS. 6B and 6C)by transfection of these cells with the indicated constructs wasperformed as noted in Example 1. Cells (5×10⁵ 293T cells or 3×10⁵ HeLacells per 6-cm dishes) were transiently transfected with the cDNAs ofthe indicated proteins together with the pCMV-β-gal, using the calciumphosphate precipitation method. Each dish was transfected with 5 μg ofthe pcDNA3 construct of interest or, when transfecting two differentconstructs, 2.5 μg of each, and 1.5 μg of β-galactosidase expressionvector. Cells were rinsed 6 to 10 h after transfection and thenincubated for a further 14 h without additional treatment. Anti-CD95(Anti-Fas-R) monoclonal antibody (CH11 (Oncor (Gaithersburg, Md.)), 0.5μg.ml) and human recombinant TNFα (100 ng/ml) were applied to the cellstogether with cycloheximide (CHX, 10 μg/ml) and incubated for anadditional 4 h. Cells were then stained with5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside (X-Gal) and examined byphase contrast microscopy. In all experiments shown, death was assessed24 h after transfection for HeLa—Fas cells and 20 h after transfectionfor 293T cells. Data shown (mean±SD; n equals at least threeexperiments) are the percentage of blue cells counted that showedmembrane blebbing.

In FIG. 6D there are shown reproductions of micrographs depicting themorphology of 293-T cells transiently expressing the indicatedconstructs. Pictures were taken 20 h after transfection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one aspect, to novel G1 proteins whichare capable of binding to or interacting directly or indirectly withMORT-1 or with MORT-1-binding proteins such as, for example, Mch4 andMACH and thereby of binding to the intracellular domain of the FAS-Rreceptor, to which MORT-1 binds, or of binding to the intracellulardomain of the p55 TNF-R, to which the protein TRADD binds and to whichTRADD protein MORT-1 binds. Hence, the G1 proteins of the presentinvention are considered as mediators or modulators of FAS-R, having arole in, for example, the signaling process that is initiated by thebinding of FAS ligand to FAS-R, and likewise also having a role in thesignaling process that is initiated by the binding of TNF to p55-R. Ofthe G1 proteins of the present invention are included the newlydiscovered G1 and its isoforms.

More particularly, in accordance with the present invention, a newprotein G1 (also called CASH), which is apparently a homolog of thenematode protease CED3 has been disclosed. This new G1 protein which,although being closely related, does however display some differences ofstructure and of substrate specificity, and as such may serve somewhatdifferent functions in mammalian cells. Indeed, two different activitiesof the proteases are known. The main role of ICE (also called CASP-1)seems to be the processing of the IL-1β precursor, while CED3 has beenclearly shown to serve as an effector of programmed cell death. Thislatter role also appears to be the role of at least some of themammalian homologs, for example some of the MACH (also called CASP-8)isoforms of the above noted co-owned co-pending patent applications, aswell as the above mentioned related Mch4 (also called CASP-10) to whichG1 of the present invention binds. The amino acid sequence of the MACHα1shows closest resemblance to CPP32 (also called CASP-3), the closestknown mammalian homolog of CED3. The substrate specificity of MACH isalso similar to that of CPP32, except that MACHα1 seems to have a morerestricted substrate specificity than that of CPP32. CPP32 cleavespreferentially the substrate peptide corresponding to a cleavage site inpoly (ADP ribose) polymerase (PARP), yet also has some proteolyticactivity against the peptide corresponding to the ICE cleavage site inthe IL-1β precursor. MACHα1 seems, however, to be solely capable ofcleaving the PARP-derived sequence. These relationships of MACHα1 toCPP32 and CED3, and its dissimilarities to ICE, are consistent with theidea that MACHα1 serves, similarly to CED3, as regulator of cell death.MACHα1 displays, though, some sequence features which distinguish itfrom CED3 and from CPP32, as well as from all other members of theCED3/ICE family. The C terminal part of MACHα1, upstream to its CED3/ICEhomology region, shows no resemblance at all to the upstream region ofany of the other homologs. There are also some unique sequence featuresto the CED3/ICE homology region of the protein. These differencessuggest that MACHα1 belongs to a distinct evolutionary branch of thefamily and that its contribution to cell death somewhat differs fromthat of the previously described CED3/ICE homologs. Likewise the G1protein of the present invention and its possible isoforms also showsome distinct differences in the CED3/ICE homology region within the G1sequence and as such these differences may represent unique featuresreflecting specificity of activity for G1 in mammalian cells.

One important difference may concern the way in which the function ofthe protease is regulated. Being involved both in developmentallyrelated cell death processes and in receptor-induced immune cytolysis,the cleavage of proteins by proteases of the CED3/ICE family should beamenable to regulation both by signals that are formed within the celland by signals emanating from cell surface receptors. In developmentalcell death processes, the activation of such proteases seems to involvemechanisms that affect gene expression, resulting in enhanced synthesisof the proteases, as well as in decreased synthesis of proteins likeBCL-2, that antagonize their apoptopic effect. This is clearly not thecase, however, for the cytotoxicity triggered by FAS-R or the TNFreceptors. Cells can be killed by TNF or the FAS-R ligand even whentheir protein synthesis activity is fully blocked (they are in factkilled more effectively then) and remain stimulus-dependent under theseconditions. Activation of proteases of the CED3/ICE family by the TNFreceptors and FAS-R may thus occur by a mechanism which isprotein-synthesis-independent. The unique sequence properties of MACHα1may allow it to take part in such a mechanism. Similarly, the uniquesequence properties of the G1 protein of the present invention may alsoendow it with an ability to take part in such a mechanism.

Thus, the new G1 protein may be yet another member of the recently foundgroup of proteases including the above mentioned MACH (and its isoforms)and Mch4 which have been found to associate, either directly or throughan adapter protein, with the intracellular domain of a cell surfacereceptor. By inference from the way of action of receptor-associatedproteins that have other enzymatic activities, it seems plausible thatthe binding of G1 to Mch4 or of G1 to MACH (or isoform Machα1) and, inturn, the binding of Mch4 of Mach to MORT-1, or the direct binding of G1to MORT1 allows the stimulation of the G1 and/or Mch4 and/or the MACHprotease activity upon triggering of FAS-R by Fas ligand. It may alsoallow activation of the protease by the p55-R, through the binding ofMORT1 to TRADD, which binds to p55-R.

Other members of the CED3/ICE family were found to exhibit full activityonly after proteolytic processing, which occurs either by theirself-cleavage or by effects of other proteases of this family (reviewedin Kumas, 1995; Henkart, 1996). For example, as detailed in the abovementioned co-owned and co-pending patent applications regarding MACH,the cytotoxic effect resulting from co-expression of the two majorpotential self-cleavage products of MACHα1, as opposed to the lack ofcytotoxicity in cells that express the full-length CED3/ICE homologyregion, is consistent with the possibility that also MACHα1 gains fullactivity only after its processing. The enzymatic activity observed inlysates of bacteria that express the full length region apparentlyreflect self processing of the protein produced under these conditionsor processing by some bacterial proteases. In what way this processingoccurs within the mammalian cell, and how it can be brought about bytriggering of FAS-R and p55-R, is not known, nor is it clear yet whatrelative contribution the protease activity of MACHα1 makes to theFAS-R- and and TNF-induced cytotoxicity. Evaluation of this contributionis complicated by the fact that also expression of MACHβ1, which lacksthe CED3/ICE homology region, results in marked cytotoxicity.Presumably, this cytotoxicity reflects the ability of MACHβ1 to bind toMACHα1. Due to this ability, transfected MACH molecules may impose, uponaggregation, a conformational change in the MACHα1 molecules that areendogenous to the transfected cell. Such a mechanism may well accountalso for the cytotoxicity observed when molecules that act upstream toMACH, (MORT1, TRADD or the death domains of either the p55-R or FAS-R)are over-expressed in cells. At the moment, however, one cannot excludethat the cytotoxicity observed upon induced expression of MACH or ofmolecules that act upstream to it reflect, besides the proteolyticactivity of the CED3/ICE homology region in MACH, also activation ofsome of the other mechanisms believed to take part in the FAS-R andp55-R cytotoxic effect (for example, activation of the neutral or acidsphingomyelinase). One also cannot exclude that the proteolytic activityof the CED3/ICE homology region serves other functions besidescytotoxicity induction. A clearer idea of the function of MACHα1 shouldbe gained by identification of the endogenous substrate proteins thatare cleaved upon activation of MACHα1. Finding ways to ablate theactivity of MACHα1 at will, for example by expression of inhibitorymolecules, will also contribute to understanding of the function of thisprotein, and serve as a way for regulating its activity when desired.

Hence, the G1 protein of the present invention and its possible isoformsmay behave in an analogous fashion to that mentioned for the above MACHproteins with or without direct interaction with other proteins, namely,G1 may act directly via binding to MORT-1 or may act indirectly viabinding to Mch4 and/or MACH and in turn by the binding of Mch4 and/orMACH to MORT-1 or in some other as yet not elucidated mechanism specificfor G1. Similarly, the regulation of G1 activity may be analogous tothat envisioned above for MACH protein regulation.

There may well exist within cells that express G1 natural inhibitors ofthe protease encompassed in this protein. Existence of alternativelyspliced isoforms for some of the other members of the CED3/ICE familyhas been shown to constitute a way of physiological restriction of thefunction of these proteases. Some of the isoforms of these otherproteases were reported to act as natural inhibitors of the full-lengthisoforms, apparently by forming inactive heterodimers, with them. Thismay well be the case also for G1 and some of its posible isoforms, forexample those isoforms in which the potential N-terminal cleavage siteis missing. Expression of such inhibitory isoforms may constitute amechanism of cellular self-protection against the FAS-R and TNFcytotoxicity.

G1 may have yet other functions, for example, G1 or any of its isoformsmay have an enhancing or augmenting effect on other proteins withenzymatic activity, e.g. the proteolytic activities of various Mch4 andMACH isoforms, this enhancing or augmenting activity being via amechanism whereby G1 serves to recruit other proteins to bind MORT-1(e.g. Mch4 and MACH proteins). Further, G1 or any of its isoforms mayalso serve roles not related to cytotoxicity, but rather may act asdocking sites for molecules that are involved in other non-cytotoxic,effects of FAS-R and TNF.

Some of the specific G1 isoforms in accordance with the presentinvention are exemplified in Example 1 below. One of these called G1α(CASHα) isolated from human and mouse (hG1α/hCASHα and mG1α/mCASHα,respectively) is apparently a 1 mg splice variant having a proteasehomology region and, at least in some cells (e.g. 293 cells), hascytotoxic activity. Another of these is called G1β (CASHβ) isolated fromhuman, is apparently a short splice variant without a protease homologyregion and which actually inhibits cell-death signaling pathways.

Due to the unique ability of FAS-R and the TNF receptors to cause celldeath, as well as the ability of the TNF receptors to trigger variousother tissue-damaging activities, aberration of the function of thesereceptors can be particularly deleterious to the organism. Indeed, bothexcessive and deficient function of these receptors have been shown tocontribute to the pathological manifestations of various diseases.Identifying molecules that take part in the signaling activity of thesereceptors, and finding ways to modulate the function of these molecules,constitutes a potential clue for new therapeutical approaches to thesediseases. In view of the suspected important role of G1 in FAS-R and TNFtoxicity, it seems particularly important to design drugs that can blockthe proteolytic function of this molecule, as has been done for someother members of the CED3/ICE family. The unique sequence features ofthe CED3/ICE homolog encompassed in the G1 molecules may allow designingdrugs that can affect its protection from excessive immune-mediatedcytotoxicity without interfering with physiological cell deathprocesses, in which other members of the CED3/ICE family are involved.

As mentioned above, G1 or any of its isoforms may also be involved inthe modulation of other intracellular signaling pathways, such as, forexample, of other cytotoxic mediators or inducers, or other proteins ina MORT-1 or MORT-1-binding protein-independent fashion. Further, G1 orat least its isoforms which have a protease-like region without actualprotease activity may be involved in primarily an inhibitory function,namely, inhibiting those pathways, e.g. signaling pathways in general orcytotoxic pathways in particular, in which G1 or its isoforms areinvolved either by binding directly to members of these pathways or bybinding indirectly to other proteins, which, in turn, bind to members ofthese pathways.

Thus, the present invention also concerns the DNA sequence encoding a G1protein and the G1 proteins encoded by the DNA sequences.

Moreover, the present invention further concerns the DNA sequencesencoding biologically active analogs, fragments and derivatives of theG1 protein, and the analogs, fragments and derivatives encoded thereby.The preparation of such analogs, fragments and derivatives is bystandard procedure (see for example, Sambrook et al., 1989) in which inthe DNA sequences encoding the G1 protein, one or more codons may bedeleted, added or substituted by another, to yield analogs having atleast one amino acid residue change with respect to the native protein.

A polypeptide or protein “substantially corresponding” to G1 proteinincludes not only G1 protein but also polypeptides or proteins that areanalogs of G1.

Analogs that substantially correspond to G1 protein are thosepolypeptides in which one or more amino acid of the G1 protein's aminoacid sequence has been replaced with another amino acid, deleted and/orinserted, provided that the resulting protein exhibits substantially thesame or higher biological activity as the G1 protein to which itcorresponds.

In order to substantially correspond to G1 protein, the changes in thesequence of G1 proteins, such as isoforms are generally relativelyminor. Although the number of changes may be more than ten, preferablythere are no more than ten changes, more preferably no more than five,and most preferably no more than three such changes. While any techniquecan be used to find potentially biologically active proteins whichsubstantially correspond to G1 proteins, one such technique is the useof conventional mutagenesis techniques on the DNA encoding the protein,resulting in a few modifications. The proteins expressed by such clonescan, then be screened for their ability to bind to variousMORT-1-binding proteins, such as, for example, Mch4 and MACH, or evendirectly to MORT-1, and/or FAS-R and p55-R mediating activity, and/or tomediating activity of any other intracellular pathway in ways notedabove.

“Conservative” changes are those changes which would not be expected tochange the activity of the protein and are usually the first to bescreened as these would not be expected to substantially change thesize, charge or configuration of the protein and thus would not beexpected to change the biological properties thereof.

Conservative substitutions of G1 proteins include an analog wherein atleast one amino acid residue in the polypeptide has been conservativelyreplaced by a different amino acid. Such substitutions preferably aremade in accordance with the following list as presented in Table IA,which substitutions may be determined by routine experimentation toprovide modified structural and functional properties of a synthesizedpolypeptide molecule while maintaining the biological activitycharacteristic of G1 protein. TABLE IA Original Exemplary ResidueSubstitution Ala Gly; Ser Arg Lys Asn Gln; His Asp Glu Cys Ser Gln AsnGlu Asp Gly Ala; Pro His Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg;Gln; Glu Met Leu; Tyr; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr TyrTrp; Phe Val Ile; Leu

Alternatively, another group of substitutions of G1 protein are those inwhich at least one amino acid residue in the polypeptide has beenremoved and a different residue inserted in its place according to thefollowing Table IB. The types of substitutions which may be made in thepolypeptide may be based on analysis of the frequencies of amino acidchanges between a homologous protein of different species, such as thosepresented in Table 1-2 of Schulz et al., G. E., Principles of ProteinStructure Springer-Verlag, New York, N.Y., 1798, and FIGS. 3-9 ofCreighton, T. E., Proteins: Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, Calif. 1983. Based on such an analysis,alternative conservative substitutions are defined herein as exchangeswithin one of the following five groups: TABLE IB 1. Small aliphatic,nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly); 2. Polarnegatively charged residues and their amides: Asp, Asn, Glu, Gln; 3.Polar, positively charged residues: His, Arg, Lys; 4. Large aliphaticnonpolar residues: Met, Leu, Ile, Val (Cys); and 5. Large aromaticresidues: Phe, Tyr, Trp.

The three amino acid residues in parentheses above have special roles inprotein architecture. Gly is the only residue lacking any side chain andthus imparts flexibility to the chain. This however tends to promote theformation of secondary structure other than a-helical. Pro, because ofits unusual geometry, tightly constrains the chain and generally tendsto promote β-turn-like structures, although in some cases Cys can becapable of participating in disulfide bond formation which is importantin protein folding. Note that Schulz et al., supra, would merge Groups 1and 2, above. Note also that Tyr, because of its hydrogen bondingpotential, has significant kinship with Ser, and Thr, etc.

Conservative amino acid substitutions according to the presentinvention, e.g., as presented above, are known in the art and would beexpected to maintain biological and structural properties of thepolypeptide after amino acid substitution. Most deletions andsubstitutions according to the present invention are those which do notproduce radical changes in the characteristics of the protein orpolypeptide molecule. “Characteristics” is defined in a non-inclusivemanner to define both changes in secondary structure, e.g. a-helix orβ-sheet, as well as changes in biological activity, e.g., binding ofMORT-1-binding proteins or of MORT-1 or mediation of FAS-R ligand or TNFeffect on cells.

Examples of production of amino acid substitutions in proteins which canbe used for obtaining analogs of G1 proteins for use in the presentinvention include any known method steps, such as presented in U.S.patent RE 33,653, U.S. Pat. Nos. 4,959,314, 4,588,585 and 4,737,462, toMark et al.; U.S. Pat. No. 5,116,943 to Koths et al., U.S. Pat. No.4,965,195 to Namen et al.; U.S. Pat. No. 4,879,111 to Chong et al.; andU.S. Pat. No. 5,017,691 to Lee et al.; and lysine substituted proteinspresented in U.S. Pat. No. 4,904,584 (Shaw et al.).

Besides conservative substitutions discussed above which would notsignificantly change the activity of G1 protein, either conservativesubstitutions or less conservative and more random changes, which leadto an increase in biological activity of the analogs of G1 proteins, areintended to be within the scope of the invention.

When the exact effect of the substitution or deletion is to beconfirmed, one skilled in the art will appreciate that the effect of thesubstitution(s), deletion(s), etc., will be evaluated by routine bindingand cell death assays. Screening using such a standard test does notinvolve undue experimentation.

Acceptable analogs are those which retain at least the capability ofbinding to MORT-1-binding proteins as noted above or binding to MORT-1or other proteins, and thereby, as noted above mediate the activity ofthe FAS-R and p55-R or other proteins as noted above. In such a way,analogs can be produced which have a so-called dominant-negative effect,namely, an analog which is defective either in binding to MORT-1-bindingproteins (e.g. Mch4 or MACH) or binding to MORT-1, or other proteins, orin subsequent signaling or protease activity following such binding.Such analogs can be used, for example, to inhibit the FAS-ligand- effector effect of other proteins by competing with the natural MORT-1-bindingproteins or other proteins. For example, it appears likely that the MACHisoforms, MACHα2 and MACHα3 are “natural” analogs which serve to inhibitMACH activity by competing with the binding of the active (protease)MACH isoforms to MORT-1 which appears to be essential for the activationof these MACH isoforms. Once the active MACH isoforms cannot bind toMORT-1, the intracellular signaling pathways mediated by FAS-R and p55-Rwill thereby also be inhibited. In a similar fashion G1 or any of itsisoforms may also serve as inhibitors of the FAS-ligand effect bycompeting with various of the MORT-1-binding proteins to bind MORT-1 orby interacting with them in a way that prevents their binding to MORT-1.Likewise, so-called dominant-positive G1 analogs may be produced whichwould serve to enhance the FAS ligand or TNF effect. These would havethe same or better ability to bind MORT-1-binding proteins or even thesame or better MORT-1-binding properties and the same or bettersignaling properties of the natural G1 proteins.

At the genetic level, these analogs are generally prepared bysite-directed mutagenesis of nucleotides in the DNA encoding the G1protein, thereby producing DNA encoding the analog, and thereaftersynthesizing the DNA and expressing the polypeptide in recombinant cellculture. The analogs typically exhibit the same or increased qualitativebiological activity as the naturally occurring protein, Ausubel et al.,Current Protocols in Molecular Biology, Greene Publications and WileyInterscience, New York, N.Y., 1987-1995; Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., 1989.

Preparation of a G1 protein in accordance herewith, or an alternativenucleotide sequence encoding the same polypeptide but differing from thenatural sequence due to changes permitted by the known degeneracy of thegenetic code, can be achieved by site-specific mutagenesis of DNA thatencodes an earlier prepared analog or a native version of a G1 protein.Site-specific mutagenesis allows the production of analogs through theuse of specific oligonucleotide sequences that encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 20 to 25nucleotides in length is preferred, with about 5 to 10 complementingnucle6tides on each side of the sequence being altered. In general, thetechnique of site-specific mutagenesis is well known in the art, asexemplified by publications such as Adelman et al., DNA 2:183 (1983),the disclosure of which is incorporated herein by reference.

As will be appreciated, the site-specific mutagenesis techniquetypically employs a phage vector that exists in both a single-strandedand double-stranded form. Typical vectors useful in site-directedmutagenesis include vectors such as the M13 phage, for example, asdisclosed by Messing et al., Third Cleveland Symposium on Macromoleculesand Recombinant DNA, Editor A. Walton, Elsevier, Amsterdam (1981), thedisclosure of which is incorporated herein by reference. These phage arereadily available commercially and their use is generally well known tothose skilled in the art. Alternatively, plasmid vectors that contain asingle-stranded phage origin of replication (Veira et al., Meth.Enzymol. 153:3, 1987) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector that includeswithin its sequence a DNA sequence that encodes the relevantpolypeptide. An oligonucleotide primer bearing the desired mutatedsequence is prepared synthetically by automated DNA/oligonucleotidesynthesis. This primer is then annealed with the single-strandedprotein-sequence-containing vector, and subjected to DNA-polymerizingenzymes such as E. coli polymerase I Klenow fragment, to complete thesynthesis of the mutation-bearing strand. Thus, a mutated sequence andthe second strand bears the desired mutation. This heteroduplex vectoris then used to transform appropriate cells, such as E. coli JM101cells, and clones are selected that include recombinant vectors bearingthe mutated sequence arrangement.

After such a clone is selected, the mutated G1 protein may be removedand placed in an appropriate vector, generally a transfer or expressionvector of the type that may be employed for transfection of anappropriate host.

Accordingly, a gene or nucleic acid encoding for a G1 protein can alsobe detected, obtained and/or modified, in vitro, in situ and/or in vivo,by the use of known DNA or RNA amplification techniques, such as PCR andchemical oligonucleotide synthesis. PCR allows for the amplification(increase in number) of specific DNA sequences by repeated DNApolymerase reactions. This reaction can be used as a replacement forcloning; all that is required is a knowledge of the nucleic acidsequence. In order to carry out PCR, primers are designed which arecomplementary to the sequence of interest. The primers are thengenerated by automated DNA synthesis. Because primers can be designed tohybridize to any part of the gene, conditions can be created such thatmismatches in complementary base pairing can be tolerated. Amplificationof these mismatched regions can lead to the synthesis of a mutagenizedproduct resulting in the generation of a peptide with new properties(i.e., site directed mutagenesis). See also, e.g., Ausubel, supra, Ch.16. Also, by coupling complementary DNA (cDNA) synthesis, using reversetranscriptase, with PCR, RNA can be used as the starting material forthe synthesis of the extracellular domain of a prolactin receptorwithout cloning.

Furthermore, PCR primers can be designed to incorporate new restrictionsites or other features such as termination codons at the ends of thegene segment to be amplified. This placement of restriction sites at the5′ and 3′ ends of the amplified gene sequence allows for gene segmentsencoding G1 protein or a fragment thereof to be custom designed forligation to other sequences and/or cloning sites in vectors.

PCR and other methods of amplification of RNA and/or DNA are well knownin the art and can be used according to the present invention withoutundue; experimentation, based on the teaching and guidance presentedherein. Known methods of DNA or RNA amplification include, but are notlimited to polymerase chain reaction (PCR) and related amplificationprocesses (see, e.g., U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159,4,965,188, to Mullis et al.; U.S. Pat. Nos. 4,795,699 and 4,921,794 toTabor et al.; U.S. Pat. No. 5,142,033 to Innis; U.S. Pat. No. 5,122,464to Wilson et al.; U.S. Pat. No. 5,091,310 to Innis; 5,066,584 toGyllensten et al.; U.S. Pat. No. 4,889,818 to Gelfand et al.; U.S. Pat.No. 4,994,370 to Silver et al.; U.S. Pat. No. 4,766,067 to Biswas; U.S.Pat. No. 4,656,134 to Ringold; and Innis et al., eds., PCR Protocols: AGuide to Method and Applications) and RNA mediated amplification whichuses anti-sense RNA to the target sequence as a template for doublestranded DNA synthesis (U.S. Pat. No. 5,130,238 to Malek et al., withthe tradename NASBA); and immuno-PCR which combines the use of DNAamplification with antibody labeling (Ruzicka et al., Science 260:487(1993); Sano et al., Science 258:120 (1992); Sano et al., Biotechniques9:1378 (1991)), the entire contents of which patents and reference areentirely incorporated herein by reference.

In an analogous fashion, biologically active fragments of G1 proteins(e.g. those of any of the G1 or its isoforms) may be prepared as notedabove with respect to the analogs of G1 proteins. Suitable fragments ofG1 proteins are those which retain the G1 capability and which canmediate the biological activity of FAS-R and p55-R or other proteins asnoted above. Accordingly, G1 protein fragments can be prepared whichhave a dominant-negative or a dominant-positive effect as noted abovewith respect to the analogs. It should be noted that these fragmentsrepresent a special class of the analogs of the invention, namely, theyare defined portions of G1 proteins derived from the full G1 proteinsequence (e.g., from that of any one of the G1 or its isoforms), eachsuch portion or fragment having any of the above-noted desiredactivities. Such fragment may be, e.g., a peptide.

Similarly, derivatives may be prepared by standard modifications of theside groups of one or more amino acid residues of the G1 protein, itsanalogs or fragments, or by conjugation of the G1 protein, its analogsor fragments, to another molecule e.g. an antibody, enzyme, receptor,etc., as are well known in the art. Accordingly, “derivatives” as usedherein covers derivatives which may be prepared from the functionalgroups which occur as side chains on the residues or the N- orC-terminal groups, by means known in the art, and are included in theinvention. Derivatives may have chemical moieties such as carbohydrateor phosphate residues, provided such a fraction has the same or higherbiological activity as G1 proteins.

For example, derivatives may include aliphatic esters of the carboxylgroups, amides of the carboxyl groups by reaction with ammonia or withprimary or secondary amines, N-acyl derivatives or free amino groups ofthe amino acid residues formed with acyl moieties (e.g., alkanoyl orcarbocyclic aroyl groups) or O-acyl derivatives of free hydroxyl group(for example that of seryl or threonyl residues) formed with acylmoieties.

The term “derivatives” is intended to include only those derivativesthat do not change one amino acid to another of the twenty commonlyoccurring natural amino acids.

Although G1 protein is a protein or polypeptide, it is a sequence ofamino acid residues. A polypeptide consisting of a larger sequence whichincludes the entire sequence of a G1 protein, in accordance with thedefinitions herein, is intended to be included within the scope of sucha polypeptide as long as the additions do not affect the basic and novelcharacteristics of the invention, i.e., if they either retain orincrease the biological activity of G1 protein or can be cleaved toleave a protein or polypeptide having the biological activity of G1protein. Thus, for example, the present invention is intended to includefusion proteins of G1 protein with other amino acids or peptides.

The new G1 protein, their analogs, fragments and derivatives thereof,have a number of possible uses, for example:

(i) G1 protein, its analogs, fragments and derivatives thereof, may beused to mimic or enhance the function of MORT-1-binding proteins suchas, for example, Mch4 and MACH (including its various isoforms), or evenof MORT-1 itself, and hence the FAS-R ligand or TNF, or other proteins,in situations where an enhanced FAS-R ligand or TNF or other proteineffect is desired, such as in anti-tumor, anti-inflammatory, anti-HIVapplications, etc., where the FAS-R ligand- or TNF- or otherprotein-induced cytotoxicity is desired. In this case the G1 protein,its analogs, fragments or derivatives thereof, which enhance the FAS-Rligand or TNF or other protein effect, i.e., cytotoxic effect, may beintroduced to the cells by standard procedures known per se. Forexample, when the G1 protein is entirely intracellular (as suspected)and should be introduced only into the cells where the FAS-R ligand orTNF or other protein effect is desired, a system for specificintroduction of this protein into the cells is necessary. One way ofdoing this is by creating a recombinant animal virus, e.g., one derivedfrom Vaccinia, to the DNA of which the following two genes will beintroduced: the gene encoding a ligand that binds to cell surfaceproteins specifically expressed by the cells, e.g., ones such as theAIDs (HIV) virus gp120 protein which binds specifically to some cells(CD4 lymphocytes and related leukemias), or any other ligand that bindsspecifically to cells carrying a FAS-R or p55-R, such that therecombinant virus vector will be capable of binding such FAS-R- or p55-R-carrying cells; and the gene encoding the G1 protein. Thus, expressionof the cell-surface-binding protein on the surface of the virus willtarget the virus specifically to the tumor cell or other FAS-R- orp55-R- carrying cell, following which the G1 protein encoding sequence(e.g. G1α sequence) will be introduced into the cells via the virus, andonce expressed in the cells, will result in enhancement of the FAS-Rligand or TNF effect leading to the death of the tumor cells or otherFAS-R- or p55-R-carrying cells it is desired to kill. Construction ofsuch recombinant animal virus is by standard procedures (see forexample, Sambrook et al., 1989). Another possibility is to introduce thesequences of the G1 protein (e.g., any one of the G1 or its isoforms) inthe form of oligonucleotides which can be absorbed by the cells andexpressed therein.

A further way of enhancing such cell cytotoxicity would be to inhibitthe activity of G1 isoforms (e.g. G1β) which themselves are inhibitoryon cell cytotoxicity. Ways of inhibiting such G1 isoforms are numerousand include those listed under (ii) below as applied specifically toinhibit such inhibitory G1 isoforms such as, for example, G1β.

-   -   (ii) They may be used to inhibit the FAS-R ligand or TNF or        other protein effect, e.g., in cases such as tissue damage in        septic shock, graft-vs.-host rejection, or acute hepatitis, in        which it is desired to block the FAS-R ligand or TNF induced        FAS-R or p55-R intracellular signaling or other protein-mediated        signaling. In this situation, it is possible, for example, to        introduce into the cells, by standard procedures,        oligonucleotides having the anti-sense coding sequence for the        G1 protein, which would effectively block the translation of        mRNAs encoding the G1 protein and thereby block its expression        and lead to the inhibition of the FAS-R ligand-or TNF- or other        protein-effect. Such oligonucleotides may be introduced into the        cells using the above recombinant virus approach, the second        sequence carried by the virus being the oligonucleotide sequence

Further, as noted above and as exemplified below, at least one G1isoform has been isolated which is a ‘natural inhibitor’ of cellcytotoxicity, namely, G1β. Hence, such a G1 isoform may be administereddirectly to cells, or a suitable vector carrying a nucleotide sequenceencoding this isoform may be introduced into cells, so that whenexpressed in the cells this G1 isoform will serve to inhibit cellcytotoxicity.

Another possibility is to use antibodies specific for the G1 protein toinhibit its intracellular signaling activity.

Yet another way of inhibiting the FAS-R ligand or TNF effect is by therecently developed ribozyme approach. Ribozymes are catalytic RNAmolecules that specifically cleave RNAs. Ribozymes may be engineered tocleave target RNAs of choice, e.g., the mRNAs encoding the G1 protein ofthe invention. Such ribozymes would have a sequence specific for the G1protein mRNA and would be capable of interacting therewith(complementary binding) followed by cleavage of the mRNA, resulting in adecrease (or complete loss) in the expression of the G1 protein, thelevel of decreased expression being dependent upon the level of ribozymeexpression in the target cell. To introduce ribozymes into the cells ofchoice (e.g., those carrying FAS-R or p55-R), any suitable vector may beused, e.g., plasmid, animal virus (retrovirus) vectors, that are usuallyused for this purpose (see also (i) above, where the virus has, assecond sequence, a cDNA encoding the ribozyme sequence of choice). (Forreviews, methods etc. concerning ribozymes see Chen et al., 1992; Zhaoand Pick, 1993; Shore et al., 1993; Joseph and Burke, 1993; Shimayama etal., 1993; Cantor et al., 1993; Barinaga, 1993; Crisell et al., 1993 andKoizumi et al., 1993).

-   -   (iii) The G1 protein, its analogs, fragments or derivatives may        also be used to isolate, identify and clone other proteins of        the same class, i.e., those binding to FAS-R intracellular        domain or to functionally related receptors, or those binding to        the above noted MORT-1-binding proteins, or those binding to        MORT-1 and thereby to functionally related receptors such as        FAS-R and p55-R, and involved in the intracellular signaling        process. In this application the above noted yeast two-hybrid        system may be used, or there may be used a recently developed        system employing non-stringent Southern hybridization followed        by PCR cloning (Wilks et al., 1989). In the Wilks et al.        publication, there is described the identification and cloning        of two putative protein-tyrosine kinases by application of        non-stringent southern hybridization followed by cloning by PCR        based on the known sequence of the kinase motif, a conceived        kinase sequence. This approach may be used, in accordance with        the present invention using the sequence of the G1 protein to        identify and clone those of related MORT-1-binding proteins.    -   (iv) Yet another approach to utilizing the G1 protein, or its        analogs, fragments or derivatives thereof, of the invention is        to use them in methods of affinity chromatography to isolate and        identify other proteins or factors to which they are capable of        binding, e.g., MORT-1, MORT-1-binding proteins, or other        proteins or factors involved in the intracellular signaling        process. In this application, the G1 protein, its analogs,        fragments or derivatives thereof, of the present invention, may        be individually attached to affinity chromatography matrices and        then brought into contact with cell extracts or isolated        proteins or factors suspected of being involved in the        intracellular signaling process. Following the affinity        chromatography procedure, the other proteins or factors which        bind to the G1 protein, or its analogs, fragments or derivatives        thereof of the invention, can be eluted, isolated and        characterized.    -   (v) As noted above, the G1 protein, or its analogs, fragments or        derivatives thereof, of the invention may also be used as        immunogens (antigens) to produce specific antibodies thereto.        These antibodies may also be used for the purposes of        purification of the G1 protein (e.g., G1 or any of its isoforms)        either from cell extracts or from transformed cell lines        producing G1 protein, or its analogs or fragments. Further,        these antibodies may be used for diagnostic purposes for        identifying disorders related to abnormal functioning of the        FAS-R ligand or TNF system, e.g., overactive or underactive        FAS-R ligand- or TNF-induced cellular effects. Thus, should such        disorders be related to a malfunctioning intracellular signaling        system involving the MORT-1 protein, or various other, above        noted MORT-1-binding proteins or G1 protein itself, such        antibodies would serve as an important diagnostic tool.

It should also be noted that the isolation, identification andcharacterization of the G1 protein of the invention may be performedusing any of the well known standard screening procedures. For example,one of these screening procedures, the yeast two-hybrid procedure as isset forth herein below, was used to identify the MORT-1 protein andsubsequently the various MORT-1-binding proteins and the G1 protein ofthe invention. Likewise as noted above and below, other procedures maybe employed such as affinity chromatography, DNA hybridizationprocedures, etc. as are well known in the art, to isolate, identify andcharacterize the G1 protein of the invention or to isolate, identify andcharacterize additional proteins, factors, receptors, etc. which arecapable of binding to the G1 proteins of the invention.

As set forth hereinabove, the G1 protein may be used to generateantibodies specific to G1 proteins, e.g., G1 and its isoforms. Theseantibodies or fragments thereof may be used as set forth hereinbelow indetail, it being understood that in these applications the antibodies orfragments thereof are those specific for G1 proteins.

Based on the findings in accordance with the present invention that atleast some of the G1 or its possible isoforms are proteases related tothe proteases of the CED3/ICE family of proteases, the followingspecific medical applications are envisioned for these G1 proteins andisoforms: it has been found that specific inhibitors of other CED3/ICEproteases, some of which are cell permeable, already exist, which canblock effectively programmed cell death processes. Hence, it is possiblein accordance with the present invention to design inhibitors that canprevent FAS-R ligand- or TNF-induced cell death, the pathways in whichthe G1 protease isoforms are involved. Further, in view of the uniquesequence features of these new G1 proteases, it seems possible to designinhibitors that will be highly specific to the TNF- and FAS-Rligand-induced effects. The findings of the present invention alsoprovide a way to study the mechanism in which the “killing protease” isactivated in response to FAS-R ligand and TNF, this allowing subsequentdevelopment of drugs that can control the extent of this activation.There are many diseases in which such drugs can be of great help.Amongst others, acute hepatitis in which the acute damage to the liverseems to reflect FAS-R ligand-mediated death of the liver cells;autoimmune-induced cell death such as the death of the β Langerhanscells of the pancreas, that results in diabetes; the death of cells ingraft rejection (e.g., kidney, heart and liver); the death ofoligodendrocytes in the brain in multiple sclerosis; and AIDS-inhibitedT cell suicide which causes proliferation of the AIDS virus and hencethe AIDS disease.

As mentioned hereinabove, it is possible that G1 or one or more of itspossible isoforms (e.g. the G1β isoform) may serve as “natural”inhibitors of the G1 protease or G1 protease isoforms, and these maythus be employed as the above noted specific inhibitors of these G1proteases. Likewise, other substances such as peptides, organiccompounds, antibodies, etc. may also be screened to obtain specificdrugs which are capable of inhibiting the G1 proteases.

A non-limiting example of how peptide inhibitors of the G1 proteaseswould be designed and screened is based on previous studies on peptideinhibitors of ICE or ICE-like proteases, the substrate specificity ofICE and strategies for epitope analysis using peptide synthesis. Theminimum requirement for efficient cleavage of peptide by ICE was foundto involve four amino acids to the left of the cleavage site with astrong preference for aspartic acid in the P₁ position and withmethylamine being sufficient to the right of the P₁ position (Sleath etal., 1990; Howard et al., 1991; Thornberry et al., 1992). Furthermore,the fluorogenic substrate peptide (a tetrapeptide),acetyl-Asp-Glu-Val-Asp-a-(4-methyl-coumaryl-7-amide) abbreviatedAc-DEVD-AMC (SEQ ID NO:10), corresponds to a sequence in poly(ADP-ribose) polymerase (PARP) found to be cleaved in cells shortlyafter FAS-R stimulation, as well as other apoptopic processes (Kaufmann,1989; Kaufmann et al., 1993; Lazebnik et al., 1994), and is cleavedeffectively by CPP32 (a member of the CED3/ICE protease family) and MACHproteases (and likewise also possibly by G1 proteases).

As Asp in the P₁ position of the substrate appears to be important,tetrapeptides having Asp as the fourth amino acid residue amd variouscombinations of amino acids in the first three residue positions can berapidly screened for binding to the active site of G1 proteases using,for example, the method developed by Geysen (Geysen, 1985; Geysen etal., 1987) where a large number of peptides on solid supports werescreened for specific interactions with antibodies. The binding of MACHproteases to specific peptides can be detected by a variety of wellknown detection methods within the skill of those in the art, such asradiolabeling of the G1 proteases, etc. This method of Geysen's wasshown to be capable of testing at least 4000 peptides each working day.

Since it may be advantageous to design peptide inhibitors thatselectively inhibit G1 proteases without interfering with physiologicalcell death processes in which other members of the CED3/ICE family ofproteases are involved, the pool of peptides binding to G1 proteases inan assay such as the one described above can be further synthesized as afluorogenic substrate peptide to test for selective cleavage by G1proteases without being cleaved by other CED3/ICE proteases. Peptideswhich are determined to be selectively cleaved by the G1 proteases, canthen be modified to enhance cell permeability and inhibit the cell deathactivity of G1 either reversibly or irreversibly. Thornberry et al.(1994) reported that a tetrapeptide (acyloxy) methyl ketoneAc-Tyr-Val-Ala-Asp-CH₂OC (O)-[2,6-(CF₃)₂] Ph (SEQ ID NO:11) was a potentinactivator of ICE. Similarly, Milligan et al. (1995) reported thattetrapeptide inhibitors having a chloromethylketone (irreversibly) oraldehyde (reversibly) groups inhibited ICE. In addition, abenzyloxycarboxyl-Asp-CH₂OC (O)-2,6-dichlorobenzene (DCB) was shown toinhibit ICE (Mashima et al., 1995). Accordingly, tetrapeptides thatselectively bind to G1 proteases can be modified with, for example, analdehyde group, chloromethylketone, (acyloxy) methyl ketone or a CH₂OC(O)-DCB group to create a peptide inhibitor of G1 protease activity.

While some specific inhibitors of other CED3/ICE proteases are cellpermeable, the cell permeability of peptide inhibitors may need to beenhanced. For instance, peptides can be chemically modified orderivatized to enhance their permeability across the cell membrane andfacilitate the transport of such peptides through the membrane and intothe cytoplasm. Muranishi et al. (1991) reported derivatizingthyrotropin-releasing hormone with lauric acid to form a lipophiliclauroyl derivative with good penetration characteristics across cellmembranes. Zacharia et al. (1991) also reported the oxidation ofmethionine to sulfoxide and the replacement of the peptide bond with itsketomethylene isoester (COCH₂) to facilitate transport of peptidesthrough the cell membrane. These are just some of the knownmodifications and derivatives that are well within the skill of those inthe art.

Furthermore, drug or peptide inhibitors, which are capable of inhibitingthe cell death activity of G1 or its possible isoforms can be conjugatedor complexed with molecules that facilitate entry into the cell.

U.S. Pat. No. 5,149,782 discloses conjugating a molecule to betransported across the cell membrane with a membrane blending agent suchas fusogenic polypeptides, ion-channel forming polypeptides, othermembrane polypeptides, and long chain fatty acids, e.g. myristic acid,palmitic acid. These membrane blending agents insert the molecularconjugates into the lipid bilayer of cellular membranes and facilitatetheir entry into the cytoplasm.

Low et al., U.S. Pat. No. 5,108,921, reviews available methods fortransmembrane delivery of molecules such as, but not limited to,proteins and nucleic acids by the mechanism of receptor mediatedendocytotic activity. These receptor systems include those recognizinggalactose, mannose, mannose 6-phosphate, transferrin,asialoglycoprotein, transcobalamin (vitamin B₁₂), α-2 macroglobulins,insulin and other peptide growth factors such as epidermal growth factor(EGF). Low et al. teaches that nutrient receptors, such as receptors forbiotin and folate, can be advantageously used to enhance transportacross the cell membrane due to the location and multiplicity of biotinand folate receptors on the membrane surfaces of most cells and theassociated receptor mediated transmembrane transport processes. Thus, acomplex formed between a compound to be delivered into the cytoplasm anda ligand, such as biotin or folate, is contacted with a cell membranebearing biotin or folate receptors to initiate the receptor mediatedtrans-membrane transport mechanism and thereby permit entry of thedesired compound into the cell.

ICE is known to have the ability to tolerate liberal substitutions inthe P₂ position and this tolerance to liberal substitutions wasexploited to develop a potent and highly selective affinity labelcontaining a biotin tag (Thornberry et al., 1994). Consequently, the P₂position as well as possibly the N-terminus of the tetrapeptideinhibitor can be modified or derivatized, such as to with the additionof a biotin molecule, to enhance the permeability of these peptideinhibitors across the cell membrane.

In addition, it is known in the art that fusing a desired peptidesequence with a leader/signal peptide sequence to create a “chimericpeptide” will enable such a “chimeric peptide” to be transported acrossthe cell membrane into the cytoplasm.

As will be appreciated by those of skill in the art of peptides, thepeptide inhibitors of G1 proteolytic activity according to the presentinvention is meant to include peptidomimetic drugs or inhibitors, whichcan also be rapidly screened for binding to G1 protease to designperhaps more stable inhibitors.

It will also be appreciated that the same means for facilitating orenhancing the transport of peptide inhibitors across cell membranes asdiscussed above are also applicable to the G1 or its isoforms themselvesas well as other peptides and proteins which exerts their effectsintracellularly.

As regards the antibodies mentioned herein throughout, the term“antibody” is meant to include polyclonal antibodies, monoclonalantibodies (mAbs), chimeric antibodies, anti-idiotypic (anti-Id)antibodies to antibodies that can be labeled in soluble or bound form,as well as fragments thereof provided by any known technique, such as,but not limited to enzymatic cleavage, peptide synthesis or recombinanttechniques.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen. Amonoclonal antibody contains a substantially homogeneous population ofantibodies specific to antigens, which populations containssubstantially similar epitope binding sites. MAbs may be obtained bymethods known to those skilled in the art. See, for example Kohler andMilstein, Nature, 256:495-497 (1975); U.S. Pat. No. 4,376,110; Ausubelet al., eds., Harlow and Lane ANTIBODIES: A LABORATORY MANUAL, ColdSpring Harbor Laboratory (1988); and Colligan et al., eds., CurrentProtocols in Immunology, Greene Publishing Assoc. and Wiley InterscienceN.Y., (1992-1996), the contents of which references are incorporatedentirely herein by reference. Such antibodies may be of anyimmunoglobulin class including IgG, IgM, IgE, IgA, GILD and any subclassthereof. A hybridoma producing a mAb of the present invention may becultivated in vitro, in situ or in vivo. Production of high titers ofmAbs in vivo or in situ makes this the presently preferred method ofproduction.

Chimeric antibodies are molecules of which different portions arederived from different animal species, such as those having the variableregion derived from a murine mAb and a human immunoglobulin constantregion. Chimeric antibodies are primarily used to reduce immunogenicityin application and to increase yields in production, for example, wheremurine mAbs have higher yields from hybridomas but higher immunogenicityin humans, such that human/murine chimeric mAbs are used. Chimericantibodies and methods for their production are known in the art(Cabilly et al., Proc. Natl. Acad. Sci. USA 81:3273-3277 (1984);Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984);Boulianne et al., Nature 312:643-646 (1984); Cabilly et al., EuropeanPatent Application 125023 (published Nov. 14, 1984); Neuberger et al.,Nature 314:268-270 (1985); Taniguchi et al., European Patent Application171496. (published Feb. 19, 1985); Morrison et al., European PatentApplication 173494 (published Mar. 5, 1986); Neuberger et al., PCTApplication WO 8601533, (published Mar. 13, 1986); Kudo et al., EuropeanPatent Application 184187 (published Jun. 11, 1986); Sahagan et al., J.Immunol. 137:1066-1074 (1986); Robinson et al., International PatentApplication No. WO8702671 (published May 7, 1987); Liu et al., Proc.Natl. Acad. Sci USA 84:3439-3443 (1987); Sun et al., Proc. Natl. Acad.Sci USA 84:214-218 (1987); Better et al., Science 240:1041-1043 (1988);and Harlow and Lane, ANTIBODIES:A LABORATORY MANUAL, supra. Thesereferences are entirely incorporated herein by reference.

An anti-idiotypic (anti-Id) antibody is an antibody which recognizesunique determinants generally associated with the antigen-binding siteof an antibody. An Id antibody can be prepared by immunizing an animalof the same species and genetic type (e.g. mouse strain) as the sourceof the mAb to which an anti-Id is being prepared. The immunized animalwill recognize and respond to the idiotypic determinants of theimmunizing antibody by producing an antibody to these idiotypicdeterminants (the anti-Id antibody). See, for example, U.S. Pat. No.4,699,880, which is herein entirely incorporated by reference.

The anti-Id antibody may also be used as an “immunogen” to induce animmune response in yet another animal, producing a so-calledanti-anti-Id antibody. The anti-anti-Id may be epitopically identical tothe original mAb which induced the anti-Id. Thus, by using antibodies tothe idiotypic determinants of a mAb, it is possible to identify otherclones expressing antibodies of identical specificity.

Accordingly, mAbs generated against the G1 proteins, analogs, fragmentsor derivatives thereof, of the present invention may be used to induceanti-Id antibodies in suitable animals, such as BALB/c mice. Spleencells from such immunized mice are used to produce anti-Id hybridomassecreting anti-Id mAbs. Further, the anti-Id mAbs can be coupled to acarrier such as keyhole limpet hemocyanin (KLH) and used to immunizeadditional BALB/c mice. Sera from these mice will contain anti-anti-Idantibodies that have the binding properties of the original mAb specificfor an epitope of the above G1 protein, or analogs, fragments andderivatives thereof.

The anti-Id mAbs thus have their own idiotypic epitopes, or “idiotopes”structurally similar to the epitope being evaluated, such as GRBprotein-a.

The term “antibody” is also meant to include both intact molecules aswell as fragments thereof, such as, for example, Fab and F(ab′)2, whichare capable of binding antigen. Fab and F(ab′)2 fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding than an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies useful in the present invention may be used for the detectionand quantitation of the G1 protein according to the methods disclosedherein for intact antibody molecules. Such fragments are typicallyproduced by proteolytic cleavage, using enzymes such as papain (toproduce Fab fragments) or pepsin (to produce F(ab′)2 fragments).

An antibody is said to be “capable of binding” a molecule if it iscapable of specifically reacting with the molecule to thereby bind themolecule to the antibody. The term “epitope” is meant to refer to thatportion of any molecule capable of being bound by an antibody which canalso be recognized by that antibody. Epitopes or “antigenicdeterminants” usually consist of chemically active surface groupings ofmolecules such as amino acids or sugar side chains and have specificthree dimensional structural characteristics as well as specific chargecharacteristics.

An “antigen” is a molecule or a portion of a molecule capable of beingbound by an antibody which is additionally capable of inducing an animalto produce antibody capable of binding to an epitope of that antigen. Anantigen may have one or more than one epitope. The specific reactionreferred to above is meant to indicate that the antigen will react, in ahighly selective manner, with its corresponding antibody and not withthe multitude of other antibodies which may be evoked by other antigens.

The antibodies, including fragments of antibodies, useful in the presentinvention may be used to quantitatively or qualitatively detect the G1protein in a sample or to detect presence of cells which express the G1protein of the present invention. This can be accomplished byimmunofluorescence techniques employing a fluorescently labeled antibody(see below) coupled with light microscopic, flow cytometric, orfluorometric detection.

The antibodies (or fragments thereof) useful in the present inventionmay be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of the G1 protein ofthe present invention. In situ detection may be accomplished by removinga histological specimen from a patient, and providing the labeledantibody of the present invention to such a specimen. The antibody (orfragment) is preferably provided by applying or by overlaying thelabeled antibody (or fragment) to a biological sample. Through the useof such a procedure, it is possible to determine not only the presenceof the G1 protein, but also its distribution on the examined tissue.Using the present invention, those of ordinary skill will readilyperceive that any of wide variety of histological methods (such asstaining procedures) can be modified in order to achieve such in situdetection.

Such assays for the G1 protein of the present invention typicallycomprises incubating a biological sample, such as a biological fluid, atissue extract, freshly harvested cells such as lymphocytes orleukocytes, or cells which have been incubated in tissue culture, in thepresence of a detectably labeled antibody capable of identifying the G1protein, and detecting the antibody by any of a number of techniqueswell known in the art.

The biological sample may be treated with a solid phase support orcarrier such as nitrocellulose, or other solid support or carrier whichis capable of immobilizing cells, cell particles or soluble proteins.The support or carrier may then be washed with suitable buffers followedby treatment with a detectably labeled antibody in accordance with thepresent invention, as noted above. The solid phase support or carriermay then be washed with the buffer a second time to remove unboundantibody. The amount of bound label on said solid support or carrier maythen be detected by conventional means.

By “solid phase support”, “solid phase carrier”, “solid support”, “solidcarrier”, “support” or “carrier” is intended any support or carriercapable of binding antigen or antibodies. Well-known supports orcarriers, include glass, polystyrene, polypropylene, polyethylene,dextran, nylon amylases, natural and modified celluloses,polyacrylamides, gabbros and magnetite. The nature of the carrier can beeither soluble to some extent or insoluble for the purposes of thepresent invention. The support material may have virtually any possiblestructural configuration so long as the coupled molecule is capable ofbinding to an antigen or antibody. Thus, the support or carrierconfiguration may be spherical, as in a bead, cylindrical, as in theinside surface of a test tube, or the external surface of a rod.Alternatively, the surface may be flat such as a sheet, test strip, etc.Preferred supports or carriers include polystyrene beads. Those skilledin the art will know may other suitable carriers for binding antibody orantigen, or will be able to ascertain the same by use of routineexperimentation.

The binding activity of a given lot of antibody, of the invention asnoted above, may be determined according to well known methods. Thoseskilled in the art will be able to determine operative and optimal assayconditions for each determination by employing routine experimentation.

Other such steps as washing, stirring, shaking, filtering and the likemay be added to the assays as is customary or necessary for theparticular situation.

One of the ways in which an antibody in accordance with the presentinvention can be detectably labeled is by linking the same to an enzymeand used in an enzyme immunoassay (EIA). This enzyme, in turn, whenlater exposed to an appropriate substrate, will react with the substratein such a manner as to produce a chemical moiety which can be detected,for example, by spectrophotometric, fluorometric or by visual means.Enzymes which can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomeras, yeast alcohol dehydrogenase,alpha-glycerophosphate dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholin-esterase. The detection can be accomplished by colorimetricmethods which employ a chromogenic substrate for the enzyme. Detectionmay also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection may be accomplished using any of a variety of otherimmunoassays. For example, by radioactive labeling the antibodies orantibody fragments, it is possible to detect R-PTPase through the use ofa radioimmunoassay (RIA). A good description of RIA may be found inLaboratory Techniques and Biochemistry in Molecular Biology, by Work, T.S. et al., North Holland Publishing Company, NY (1978) with particularreference to the chapter entitled “An Introduction to Radioimmune Assayand Related Techniques” by Chard, T., incorporated by reference herein.The radioactive isotope can be detected by such means as the use of a gcounter or a scintillation counter or by autoradiography.

It is also possible to label an antibody in accordance with the presentinvention with a fluorescent compound. When the fluorescently labeledantibody is exposed to light of the proper wavelength, its presence canbe then detected due to fluorescence. Among the most commonly usedfluorescent labeling compounds are fluorescein isothiocyanate,rhodamine, phycoerythrine, pycocyanin, allophycocyanin, o-phthaldehydeand fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriamine pentaacetic acid (ETPA).

The antibody can also be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

An antibody molecule of the present invention may be adapted forutilization in an immunometric assay, also known as a “two-site” or“sandwich” assay. In a typical immunometric assay, a quantity ofunlabeled antibody (or fragment of antibody) is bound to a solid supportor carrier and a quantity of detectably labeled soluble antibody isadded to permit detection and/or quantitation of the ternary complexformed between solid-phase antibody, antigen, and labeled antibody.

Typical, and preferred, immunometric assays include “forward” assays inwhich the antibody bound to the solid phase is first contacted with thesample being tested to extract the antigen from the sample by formationof a binary solid phase antibody-antigen complex. After a suitableincubation period, the solid support or carrier is washed to remove theresidue of the fluid sample, including unreacted antigen, if any, andthen contacted with the solution containing an unknown quantity oflabeled antibody (which functions as a “reporter molecule”). After asecond incubation period to permit the labeled antibody to complex withthe antigen bound to the solid support or carrier through the unlabeledantibody, the solid support or carrier is washed a second time to removethe unreacted labeled antibody.

In another type of “sandwich” assay, which may also be useful with theantigens of the present invention, the so-called “simultaneous” and“reverse” assays are used. A simultaneous assay involves a singleincubation step as the antibody bound to the solid support or carrierand labeled antibody are both added to the sample being tested at thesame time. After the incubation is completed, the solid support orcarrier is washed to remove the residue of fluid sample and uncomplexedlabeled antibody. The presence of labeled antibody associated with thesolid support or carrier is then determined as it would be in aconventional “forward” sandwich assay.

In the “reverse” assay, stepwise addition first of a solution of labeledantibody to the fluid sample followed by the addition of unlabeledantibody bound to a solid support or carrier after a suitable incubationperiod is utilized. After a second incubation, the solid phase is washedin conventional fashion to free it of the residue of the sample beingtested and the solution of unreacted labeled antibody. The determinationof labeled antibody associated with a solid support or carrier is thendetermined as in the “simultaneous” and “forward” assays.

The G1 proteins of the invention may be produced by any standardrecombinant DNA procedure (see for example, Sambrook, et al., 1989 andAnsabel et al., 1987-1995, supra) in which suitable eukaryotic orprokaryotic host cells well known in the art are transformed byappropriate eukaryotic or prokaryotic vectors containing the sequencesencoding for the proteins. Accordingly, the present invention alsoconcerns such expression vectors and transformed hosts for theproduction of the proteins of the invention. As mentioned above, theseproteins also include their biologically active analogs, fragments andderivatives, and thus the vectors encoding them also include vectorsencoding analogs and fragments of these proteins, and the transformedhosts include those producing such analogs and fragments. Thederivatives of these proteins, produced by the transformed hosts, arethe derivatives produced by standard modification of the proteins ortheir analogs or fragments.

The present invention also relates to pharmaceutical compositionscomprising recombinant animal virus vectors encoding the G1 proteins,which vector also encodes a virus surface protein capable of bindingspecific target cell (e.g., cancer cells) surface proteins to direct theinsertion of the G1 protein sequences into the cells. Furtherpharmaceutical compositions of the invention comprises as the activeingredient (a) an oligonucleotide sequence encoding an anti-sensesequence of the G1 protein sequence, or (b) drugs that block theproteolytic activity of G1 or its isoforms.

Pharmaceutical compositions according to the present invention include asufficient amount of the active ingredient to achieve its intendedpurpose. In addition, the pharmaceutical compositions may containsuitable pharmaceutically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically and which can stabilizesuch preparations for administration to the subject in need thereof aswell known to those of skill in the art.

The G1 protein and its isoforms or isotypes are suspected to beexpressed in different tissues at markedly different levels andapparently also with different patterns of isotypes in an analogousfashion to the expression of MACH protein and its various isotypes asindicated in the above listed co-owned co-pending patent applications.These differences may possibly contribute to the tissue-specificfeatures of response to the Fas/APO1-ligand and TNF. As in the case ofother CED3/ICE homologs (Wang et al., 1994; Alnemri et al., 1995), thepresent inventors have previously shown (in the above mentioned patentapplications) that MACH isoforms that contain incomplete CED3/ICEregions (e.g., MACHα3) are found to have an inhibitory effect on theactivity of co-expressed MACHα1 or MACHα2 molecules; they are also foundto block death induction by Fas/APO1 and p55-R. Expression of suchinhibitory isoforms in cells may constitute a mechanism of cellularself-protection against Fas/APO1- and TNF-mediated cytotoxicity. Ananalogous inhibitory effect of at least some G1 isoforms is thussuspected. The wide heterogeneity of MACH isoforms, and likewise thesuspected, analogous heterogeneity of G1 isoforms, which greatly exceedsthat observed for any of the other proteases of the CED3/ICE family,should allow a particularly fine tuning of the function of the activeMACH isoforms, and by analogy also the active G1 isoforms in accordancewith the present invention.

It is also possible that some of the possible G1 isoforms serve otherfunctions. For example, the previously found (present inventors as notedabove) ability of MACHβ1 to bind to both MORT1 and MACHα1 suggests thatthis isoform could actually enhance the activity of the enzymaticallyactive isoforms. The mild cytotoxicity observed in 293-EBNA and MCF7cultures transfected with this isoform and the rather significantcytotoxic effect that it exerts in HeLa cells are likely to reflectactivation of endogenously-expressed MACHα molecules upon binding to thetransfected MACHβ1 molecules. Conceivably, some of the MACH isoformscould also act as docking sites for molecules that are involved inother, non-cytotoxic effects of Fas/APO1 and TNF receptors. Hence, in ananalogous fashion G1 and/or its isoforms may also have such enhancingactivities or serve as docking sites for other such molecules.

Due to the unique ability of Fas/APO1 and TNF receptors to cause celldeath, as well as the ability of the TNF receptors to trigger othertissue-damaging activities, aberrations in the function of thesereceptors could be particularly deleterious to the organism. Indeed,both excessive and deficient functioning of these receptors have beenshown to contribute to pathological manifestations of various diseases(Vassalli, 1992; Nagata and Golstein, 1995). Identifying the moleculesthat participate in the signaling activity of the receptors, and findingways to modulate the activity of these molecules, could direct newtherapeutic approaches. In view of the suspected central role of G1 inFas/APO1- and TNF-mediated toxicity, it seems particularly important todesign drugs that can block the possible proteolytic function of G1, aswas done for some other proteins of the CED3/ICE family (Thornberry etal., 1994; Miller et al., 1995; Mashima et al., 1995; Milligan et al.,1995; Enari et al., 1995; Los et al., 1995). The unique sequencefeatures of the CED3/ICE homolog apparently existing within G1 moleculescould permit the design of drugs that would specifically affect itsactivity. Such drugs could provide protection from excessiveimmune-mediated cytotoxicity involving G1, without interfering with thephysiological cell-death processes in which other members of theCED3/ICE family are involved.

Other aspects of the invention will be apparent from the followingexamples.

-   -   The invention will now be described in more detail in the        following non-limiting examples and the accompanying drawings.    -   It should also be noted that the procedures of:

-   i) two-hybrid screen and two-hybrid β-galactosidase expression    test; (ii) induced expression, metabolic labeling and    immunoprecipitation of proteins; (iii) in vitro binding; (iv)    assessment of the cytotoxicity; and (v) Northern and sequence    analyses, as set forth in Reference Examples 1 (see also Boldin et    al., 1995b) 2, and 3 (see also Boldin et al., 1996) below, with    respect to MORT-1 and a MORT-1 binding protein, (e.g. MACH),    respectively, are equally applicable (with some modifications) for    the corresponding isolation, cloning and characterization of G1 and    its possible isoforms of the present invention. These procedures are    thus to be construed as the full disclosure of the same procedures    used for the isolation, cloning and characterization of G1 in    accordance with the present invention, as detailed in Example 1    below. (Reference Examples 1-3 below also appear in the same or    equivalent form in the co-owned co-pending Israel Application Nos.    114,615, 114,986, 115,319, 116588, and 117,932, as well as the    corresponding PCT application No. PCT/US96/10521). Moreover, in the    above section entitled ‘Brief Description of the Drawings’ there is    also included some details of the experimental procedures carried    out in accordance with the present invention and these form part of    Example 1 below with respect to the full disclosure of the present    invention and hance should be considered together with the    disclosure in Example 1.

REFERENCE EXAMPLE 1 Cloning and Isolation of the MORT-1 Protein WhichBinds to the Intracellular Domain of the FAS-R

(i) Two-Hybrid Screen and Two-Hybrid β-Galactosidase Expression Test

To isolate proteins interacting with the intracellular domain of theFAS-R, the yeast two-hybrid system was used (Fields and Song, 1989).Briefly, this two-hybrid system is a yeast-based genetic assay to detectspecific protein-protein interactions in vivo by restoration of aeukaryotic transcriptional activator such as GALA that has two separatedomains, a DNA binding and an activation domain, which domains whenexpressed and bound together to form a restored GAL4 protein, is capableof binding to an upstream activating sequence which in turn activates apromoter that controls the expression of a reporter gene, such as lacZor HIS3, the expression of which is readily observed in the culturedcells. In this system, the genes for the candidate interacting proteinsare cloned into separate expression vectors. In one expression vector,the sequence of the one candidate protein is cloned in phase with thesequence of the GAL4 DNA-binding domain to generate a hybrid proteinwith the GAL4 DNA-binding domain, and in the other vector, the sequenceof the second candidate protein is cloned in phase with the sequence ofthe GAL4 activation domain to generate a hybrid protein with theGAL4-activation domain. The two hybrid vectors are then co-transformedinto a yeast host strain having a lacZ or HIS3 reporter gene under thecontrol of upstream GAL4 binding sites. Only those transformed hostcells (cotransformants) in which the two hybrid proteins are expressedand are capable of interacting with each other, will be capable ofexpressing the reporter gene. In the case of the lacZ reporter gene,host cells expressing this gene will become blue in color when X-gal isadded to the cultures. Hence, blue colonies are indicative of the factthat the two cloned candidate proteins are capable of interacting witheach other.

Using this two-hybrid system, the intracellular domain, FAS-IC, wascloned, separately, into the vector pGBT9 (carrying the GAL4 DNA-bindingsequence, provided by CLONTECH, USA, see below), to create fusionproteins with the GAL4 DNA-binding domain. For the cloning of FAS-R intopGBT9, a clone encoding the full-length cDNA sequence of FAS-R (WO9531544) was used from which the intracellular domain (IC) was excisedby standard procedures using various restriction enzymes and thenisolated by standard procedures and inserted into the pGBT9 vector,opened in its multiple cloning site region (MCS), with the correspondingsuitable restriction enzymes. It should be noted that the FAS-IC extendsfrom amino acid residues 175-319 of the intact FAS-R, this portioncontaining residues 175-319 being the FAS-IC inserted into the pGBT9vector.

The above hybrid (chimeric) vector was then cotransfected together witha cDNA library from human HeLa cells cloned into the pGAD GH vector,bearing the GAL4 activating domain, into the HF7c yeast host strain (allthe above-noted vectors, pGBT9 and pGAD GH carrying the HeLa cell cDNAlibrary, and the yeast strain were purchased from Clontech Laboratories,Inc., USA, as a part of MATCHMAKER Two-Hybrid System, #PT1265-1). Theco-transfected yeasts were selected for their ability to grow in mediumlacking Histidine (His⁻ medium), growing colonies being indicative ofpositive transformants. The selected yeast clones were then tested fortheir ability to express the lacZ gene, i.e., for their LACZ activity,and this by adding X-gal to the culture medium, which is catabolized toform a blue colored product by β-galactosidase, the enzyme encoded bythe lacZ gene. Thus, blue colonies are indicative of an active lacZgene. For activity of the lacZ gene, it is necessary that the GALAtranscription activator be present in an active form in the transformedclones, namely that the GAL4 DNA-binding domain encoded by the abovehybrid vector be combined properly with the GAL4 activation domainencoded by the other hybrid vector. Such a combination is only possibleif the two proteins fused to each of the GAL4 domains are capable ofstably interacting (binding) to each other. Thus, the His⁺ and blue(LACZ⁺) colonies that were isolated are colonies which have beencotransfected with a vector encoding FAS-IC and a vector encoding aprotein product of human HeLa cell origin that is capable of bindingstably to FAS-IC.

The plasmid DNA from the above His⁺, LACZ⁺ yeast colonies was isolatedand electroporated into E. coli strain HB101 by standard proceduresfollowed by selection of Leu⁺ and Ampicillin resistant transformants,these transformants being the ones carrying the hybrid pGAD GH vectorwhich has both the Amp^(R) and Leu2 coding sequences. Such transformantstherefore are clones carrying the sequences encoding newly identifiedproteins capable of binding to the FAS-IC. Plasmid DNA was then isolatedfrom these transformed E. coli and retested by:

-   -   (a) retransforming them with the original FAS-R intracellular        domain hybrid plasmid (hybrid pGTB9 carrying the FAS-IC) into        yeast strain HF7 as set forth hereinabove. As controls, vectors        carrying irrelevant protein encoding sequences, e.g., pACT-lamin        or pGBT9 alone were used for cotransformation with the        FAS-IC-binding protein (i.e., MORT-1)-encoding plasmid. The        cotransformed yeasts were then tested for growth on His⁻ medium        alone, or with different levels of 3-aminotriazole; and    -   (b) retransforming the plasmid DNA and original FAS-IC hybrid        plasmid and control plasmids described in (a) into yeast host        cells of strain SFY526 and determining the LACZ⁺ activity        (effectivity of β-gal formation, i.e., blue color formation).

The results of the above tests revealed that the pattern of growth ofcolonies in His⁻ medium was identical to the pattern of LACZ activity,as assessed by the color of the colony, i.e., His⁺ colonies were alsoLACZ⁺. Further, the LACZ activity in liquid culture (preferred cultureconditions) was assessed after transfection of the GAL4 DNA-binding andactivation-domain hybrids into the SFY526 yeast hosts which have abetter LACZ inducibility with the GAL4 transcription activator than thatof the HF7 yeast host cells.

Using the above procedure, a protein previously designated HF1, and nowreferred to as MORT-1 for “Mediator of Receptor-induced Toxicity”, wasidentified, isolated and characterized.

Furthermore, it should also be mentioned that in a number of the abovetwo-hybrid β-galactosidase expression tests, the expression ofβ-galactosidase was also assessed by a preferred filter assay. In thescreening, five of about 3×10⁶ cDNAs were found to contain the MORT-1insert. The so-isolated cloned MORT-1 cDNA inserts were then sequencedusing standard DNA sequencing procedures. The amino acid sequence ofMORT-1 was deduced from the DNA sequence (for the MORT-1 DNA and aminoacid sequences, see co-owned, co-pending Israel Application Nos.112,022, 112,692, and 114,615 and their corresponding PCT applicationNo. WO96/18641). Residue numbering in the proteins encoded by the cDNAinserts are as in the Swiss-Prot data bank. Deletion mutants wereproduced by PCR, and point mutants by oligonucleotide-directedmutagenesis (Current Protocols in Molec. Biol., 1994).

(ii) Induced Expression, Metabolic Labeling and Immunoprecipitation ofProteins

MORT-1, N-linked to the FLAG octapeptide (FLAG-MORT-1; Eastman Kodak,New Haven, Conn., USA), Fas-IC, FAS-R, p55-R, a chimera comprised of theextracellular domain of p55-R (amino acids 1-168) fused to thetransmembrane and intracellular domain of FAS-R (amino acids 153-319),and the luciferase cDNA which serves as a control, were expressed inHeLa cells. Expression was carried out using a tetracycline-controlledexpression vector, in a HeLa cell clone (HtTA-1) that expresses atetracycline-controlled transactivator (Gossen and Bujard, 1992; seealso Boldin et al., 1995). Metabolic labeling with [³⁵S]methionine and[³⁵S]cysteine (DUPONT, Wilmington, Del., USA and Amersham,Buckinghamshire, England) was performed 18 hours after transfection, bya further 4 h incubation at 37° C. in Dulbecco's modified Eagle's mediumlacking methionine and cysteine, but supplemented with 2% dialyzed fetalcalf serum. The cells were then lysed in RIPA buffer (10 mM Tris-HCl, pH7.5, 150 mM NaCl, 1% NP-40, 1% deoxycholate, 0.1% SDS and 1 mM EDTA) andthe lysate was precleared by incubation with irrelevant rabbit antiserum(3 μl/ml) and Protein G Sepharose beads (Pharmacia, Uppsala, Sweden; 60μl/ml). Immunoprecipitation was performed by 1 h incubation at 4° C. of0.3 ml aliquots of lysate with mouse monoclonal antibodies (5μl/aliquot) against the FLAG octopeptide (M2; Eastman Kodak), p55-R (#18and #20; Engelmann et al., 1990), or FAS-R (ZB4; Kamiya Southand Oaks,Calif., USA), or with isotype matched mouse antibodies as a control,followed by a further 1 h incubation with Protein G Sepharose beads (30μl/aliquot).

(iii) In Bitro Binding

Glutathione S-transferase (GST) fusions with the wild type or a mutatedFas-IC were produced and adsorbed to glutathione-agarose beads; seeBoldin et al., 1995; Current Protocols in Molecular Biology, 1994;Frangioni and Neel, 1993). Binding of metabolically-labeled FLAG-MORT-1fusion protein to GST-Fas-IC was assessed by incubating the beads for 2h at 4° C. with extracts of HeLa cells, metabolically labeled with[³⁵S]methionine (60 μCi/ml), that express FLAG-MORT-1. The extracts wereprepared in a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,0.1% NP-40, 1 mM dithiotreitol, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 20 μg/ml Aprotonin, 20 μg/ml Leupeptin, 10 mM sodium fluorideand 0.1 mM sodium vanadate (1 ml per 5×10⁵ cells).

(iv) Assessment of the Cytotoxicity Triggered by Induced Expression ofMORT-1

MORT-1, Fas-IC, p55-IC and luciferase cDNAs were inserted into atetracycline-controlled expression vector and transfected to HtTA-1cells (a HeLa cell line) (Gossen and Bujard, 1992) together with thesecreted placental alkaline phosphatase cDNA, placed under control ofSV40 promoter (the pSBC-2 vector, Dirks et al., 1993). Cell death wasassessed 40 hours after transfection, either by the neutral-red uptakeassay (Wallach, 1984) or, for assessing specifically the death in thosecells that express the transfected cDNAs, by determining the amounts ofplacental alkaline phosphatase (Berger et al., 1988) secreted to thegrowth medium at the last 5 hours of incubation.

In another set of experiments to analyze the region of the MORT-1protein involved in the binding to the FAS-IC, the following proteinswere expressed transiently in HeLa cells that contain atetracycline-controlled transactivator (HtTA-1), using atetracycline-controlled expression vector (pUHD10-3): Human FAS-R alone;Human FAS-R as well as the N-terminal part of MORT-1 (amino acids 1-117,the “MORT-1 head”); Human FAS-R as well as the C-terminal part ofMORT-1, which contains its ‘death domain’ homology region (amino acids130-245, the “MORT-1 DD”); FLAG-55.11 (amino acids 309-900 of protein55.11 fused at the N-terminus to the FLAG octapeptide, the protein 55.11being a p55-IC-specific binding protein. Twelve hours aftertransfection, the cells were trypsinized and re-seeded at aconcentration of 30,000 cells/well. After 24 hrs further incubation, thecells were treated for 6 hrs with a monoclonal antibody against theextracellular domain of FAS-R (monoclonal antibody CH-11, Oncor,Gaithersburg, Md., USA) at various concentrations (0.001-10 μg/mlmonoclonal antibody), in the presence of 10 μg/ml cycloheximide. Cellviability was then determined by the neutral-red uptake assay and theresults were presented in terms of % viable cells as compared to cellsthat had been incubated with cycloheximide alone (in the absence ofanti-FAS-R monoclonal antibody CH-11).

(v) Northern and Sequence Analyses

Poly A⁺ RNA was isolated from total RNA of HeLa cells (Oligotex-dT mRNAkit. QIAGEN, Hilden, Germany). Northern analysis using the MORT-1 cDNAas a probe was performed by conventional methods (see Boldin et al.,1995). The nucleotide sequence of MORT-1 was determined in bothdirections by the dideoxy chain termination method.

Sequence analysis of the MORT-1 cDNA cloned by the two-hybrid procedureindicated that it encodes a novel protein. Applying the two-hybrid testfurther to evaluate the specificity of the binding of this protein(MORT-1 for “Mediator of Receptor-induced Toxicity”) to Fas-IC, and todefine the particular region in Fas-IC to which it binds, led to thefollowing findings: (a) The MORT-1 protein binds both to human and tomouse Fas-IC, but not to several other tested proteins, including threereceptors of the TNF/NGF receptor family (p55 and p75 TNF receptors andCD40); (b) Replacement mutations at position 225 (Ile) in the ‘deathdomain’ of FAS-R, shown to abolish signaling both in vitro and in vivo(the lpr^(cg) mutation (Watanabe-Fukunaga et al., 1992; Itoh and Nagata,1993), also prevents binding of MORT-1 to the FAS-IC; (c) The MORT-1binding-site in FAS-R occurs within the ‘death domain’ of this receptor;and (d) MORT-1 binds to itself. This self-binding, and the binding ofMORT-1 to FAS-R involve different regions of the protein: A fragment ofMORT-1 corresponding to residues 1-117 binds to the full-length MORT-1,but does not bind to itself nor to the FAS-IC. Conversely, a fragmentcorresponding to residues 130-245 binds to FAS-R, yet does not bind toMORT-1. Furthermore, it also arose from the results that the ‘deathdomain’ region of FAS-R is critical for FAS-IC self-association, as isthe ‘death domain’ region of p55-R for p55-IC self-association. Thedeletions on both sides of these ‘death domains’ does not affect theself-association ability thereof while, however, a deletion within these‘death domains’ does affect the self-association. In the case of MORT-1,the binding of MORT-1 to FAS-IC is also dependent upon the complete(fill) ‘death domain’ of FAS-R, while however, it is also not dependenton the regions outside of the FAS-R ‘death domain’ region for FAS-ICbinding.

The interaction of the proteins encoded by the Gal4 DNA binding domainand activation-domain constructs (pGBT9 and pGAD-GH) within transfectedSFY526 yeasts was assessed by β-galactosidase expression filter assay.The DNA-binding-domain constructs included four constructs of the humanFas-IC, four constructs of the mouse Fas-IC including two full-lengthconstructs having Ile to Leu or Ile to Ala replacement mutations atposition 225 (I225N and I225A, respectively), and three MORT-1constructs. The activation-domain constructs included three MORT-1constructs, the MORT-1 portion being as in the DNA-binding-domainconstructs; and a full-length human Fas-IC construct, the Fas-IC portionbeing the same as in the above DNA-binding domain construct. Theintracellular domains of human p55 TNF receptor (p55-IC residues206-426), human CD40 (CD40-IC, residues 216-277) and human p75 TNFreceptor (p75-IC, residues 287-461) as well as lamin, cyclin D and“empty” Gal4 (pGBT9) vectors served as negative controls in the form ofDNA-binding domain constructs. SNF-1 and SNF4 served as positivecontrols in the form of DNA-binding-domain (SNF1) and activation-domain(SNF4) constructs. “Empty” Gal4 vectors (pGAD-GH) also served asnegative controls in the form of activation domain constructs. Thesymbols “++” and “+” used in the presentation of the results of theabove analysis denote the development of strong color within 30 and 90min of the assay, respectively; and “−” denotes no development of colorwithin 24 h.

Expression of MORT-1 molecules fused at their N-terminus with the FLAGoctapeptide (FLAG-MORT-1) yielded in HeLa cells proteins of fourdistinct sizes—about 27, 28, 32, and 34 kD. The interaction of MORT-1with Fas-IC in vitro was observed by performing an immunoprecipitate ofproteins from extracts of HeLa cells transfected with the FLAG-MORT-1fusion protein or with luciferase cDNA as a control, theimmunoprecipitation being performed with anti-FLAG antibody (aFLAG). Theinteraction in vitro was also demonstrated between MORT-1 and FAS-ICwherein MORT-1 is in the form of [³⁵S] methionine-metabolically labeledFLAG-MORT-1 fusion proteins obtained from extracts of transfected HeLacells and FAS-IC is in the form of human and mouse GST-FAS-IC fusionproteins including one having a replacement mutation at position 225 inFAS-IC, all of which GST-FAS-IC fusion proteins were produced in E.coli. The GST-fusion proteins were attached to glutathione beads beforeinteraction with the extracts containing the MORT-1-FLAG fusion proteinfollowing this interaction, SDS-PAGE was performed. Thus, the in vitrointeraction was evaluated by assessing, by autoradiography followingSDS-PAGE, the binding of [³⁵S] metabolically labeled MORT-1, produced intransfected HeLa cells as a fusion with the FLAG octapeptide(FLAG-MORT-1), to GST, GST fusion with the human or mouse Fas-IC(GST-huFas-IC, GST-mFas-IC) or to GST fusion with Fas-IC containing aIle to Ala replacement mutation at position 225. It was shown that allfour FLAG-MORT-1 proteins showed ability to bind to Fas-IC uponincubation with a GST-Fas-IC fusion protein. As in the yeast two-hybridtest, MORT-1 did not bind to a GST-Fas-IC fusion protein with areplacement at the lpr^(cg) mutation site (I225A).

The proteins encoded by the FLAG-MORT-1 cDNA showed also an ability tobind to the intracellular domain of FAS-R, as well as to theintracellular domain of FAS-R chimera whose extracellular domain wasreplaced with that of p55-R (p55-FAS), when co-expressed with thesereceptors in HeLa cells. In this case, interaction of MORT-1 with FAS-ICin transfected HeLa cells, i.e., in vivo, as observed withimmunoprecipitates of various transfected HeLa cells demonstrated the invivo interaction and specificity of the interaction between MORT-1 andFAS-IC in cells co-transfected with constructs encoding these proteins.Thus, FLAG-MORT-1 fusion protein was expressed and metabolically labeledwith [³⁵S] cystein (20 μCi/ml) and [³⁵S]methionine (40 μCi/ml) in HeLacells, alone, or together with human FAS-R, FAS-R chimera in which theextracellular domain of FAS-R was replaced with the corresponding regionin the human p55-R (p55-FAS), or the human p55-R, as negative control.Cross immunoprecipitation of MORT-1 with the co-expressed receptor wasperformed using various specific antibodies. The results indicated that,FLAG-MORT-1 is capable of binding to the intracellular domain of FAS-R,as well as to the intracellular domain of a FAS-R-p55-R chimera havingthe extracellular domain of p55-R and the intracellular domain of FAS-R,when co-expressed with these receptors in the HeLa cells. Further,immunoprecipitation of FLAG-MORT-1 from extracts of the transfectedcells also resulted in precipitation of the co-expressed FAS-R or theco-expressed p55-FAS chimera. Conversely, immunoprecipitation of thesereceptors resulted in the coprecipitation of the FLAG-MORT-1.

Northern analysis using the MORT-1 cDNA as probe revealed a singlehybridizing transcript in HeLa cells. In a Northern blot in which polyA⁺ RNA (0.3 μg) from transfected cells was hybridized with MORT-1 cDNA,the size of the RNA transcript (about 1.8 kb) was found to be close tothe size of the MORT-1 cDNA (about 1702 nucleotides).

In sequence analysis, the cDNA was found to contain an open readingframe of about 250 amino acids. In the above co-owned co-pendingapplications, the MORT-1 DNA and amino acid sequences are shown (seeW096/18641). In these sequences the ‘death domain’ motif is underlined,as is a possible start Met residue (position 49; bold, underlined M) andthe translation stop codon (the asterik under the codon at position769-771). This ‘death domain’ motif shares homology with the known p55-Rand FAS-R ‘death domain’ motifs (p55DD and FAS-DD). In order todetermine the precise C-terminal end of MORT-1 and to obtain evidenceconcerning the precise N-terminal (initial Met residue) end of MORT-1,additional experiments were carried out as follows:

Using the methods described above, a number of constructs encodingMORT-1 molecules fused at their N-terminus with the FLAG octapeptide(FLAG-MORT-1) were constructed and expressed in HeLa cells withmetabolic labeling of the expressed proteins using ³⁵S-cysteine and³⁵S-methionine. The MORT-1-FLAG molecules were encoded by the followingcDNAs containing different portions of the MORT-1-encoding sequence:

-   -   i) The FLAG octapeptide cDNA linked to the 5′ end of the MORT-1        cDNA from which nucleotides 1-145 were deleted;    -   ii) The FLAG octapeptide cDNA linked to the 5′ end of the MORT-1        full length cDNA;    -   iii) The FLAG octapeptide cDNA linked to the 5′ end of the        MORT-1 cDNA from which nucleotides 1-145 as well as nucleotides        832-1701 were deleted and the codon GCC at position 142-144 was        mutated to TCC to prevent start of translation at this site.

Following expression of the above FLAG-MORT-1 fusion products,immunoprecipitation was carried out as mentioned above, using eitheranti-FLAG monoclonal antibodies or as a control, anti-p75 TNF-Rantibodies, followed by SDS-PAGE (10% acrylamide) and autoradiography.The results of the analysis with the above FLAG-MORT-1 fusion productsconfirmed (validated) the C-terminal end of MORT-1 and provided evidencethat the N-terminal end of MORT-1 may be at position 49 of the sequence.

Indeed, it has been shown by additional expression experiments of MORT-1without the FLAG octapeptide fused to its 5′-end, that Met⁴⁹ serves asan effective site of translation initiation.

A search conducted in the ‘Gene Bank’ and ‘Protein Bank’ DataBasesrevealed that at the time, there was no sequence corresponding to thatof the above isolated MORT-1 sequence. Thus, MORT-1 represented a newFAS-IC-specific binding protein.

High expression of p55-IC results in triggering of a cytocidal effect(Boldin et al., 1995). The expression of Fas-IC in HeLa cells also hassuch an effect, though to a lower extent, which could be detected onlywith the use of a sensitive assay. The ligand independent triggering ofcytocidal effects in cells transfected with MORT-1, as well as humanp55-IC and FAS-IC, was thus analyzed. The effect of transient expressionof MORT-1, human Fas-IC, human p55-IC, or luciferase that served as acontrol, on the viability of HeLa cells was assessed using atetracycline-controlled expression vector. Cell viability was evaluated40 min after transfecting these cDNAs either in the presence or absenceof tetracycline (1 μg/ml, to block expression), together with a cDNAencoding the secreted placental alkaline phosphatase. Cell viability wasdetermined either by the neutral red uptake assay or, for determiningspecifically the viability of those particular cells that express thetransfected DNA, by measuring the amounts of placental alkalinephosphatase secreted to the growth medium.

The above analysis revealed that the expression of MORT-1 in HeLa cellsresulted in significant cell death, greater than that caused by FAS-ICexpression. These cytotoxic effects of all of p55-IC, FAS-IC and MORT-1seem to be related to the ‘death domain’ regions, present in all ofthese proteins, which ‘death domains’ have a propensity toself-associate, and thereby possibly prompting the cytotoxic effects.

In view of the above mentioned characteristics of MORT-1, namely, thespecific association of MORT-1 with that particular region in FAS-Rwhich is involved in cell death induction, and the fact that even aslight change of structure in that region, which prevents signaling (thelpr^(cg) mutation) abolishes also the binding of MORT-1, indicates thatthis protein plays a role in the signaling or triggering of cell death.This notion is further supported by the observed ability of MORT-1 totrigger by itself a cytocidal effect. Thus, MORT-1 may function as (i) amodulator of the self-association of FAS-R by its own ability to bind toFAS-R as well as to itself, or (ii) serve as a docking site foradditional proteins that are involved in the FAS-R signaling, i.e.,MORT-1 may be a ‘docking’ protein and may therefore bind other receptorsbesides FAS-R, or (iii) constitutes part of a distinct signaling systemthat interacts with FAS-R signaling.

In order to further analyze the regions of MORT-1 involved in FAS-ICbinding and modulation of the FAS-R-mediated cellular effects(cytotoxicity), the above-mentioned experiments were carried out, usingvectors encoding portions of MORT-1 (the ‘MORT-1 head’, amino acids1-117 and the ‘MORT-1 dd’, amino acids 130-245) (separately), with avector encoding the human FAS-R for co-transfections of HeLa cells. Inthese experiments, the various proteins and combinations of proteinswere expressed transiently in HeLa cells that contain atetracycline-controlled transactivator (HtTA-1) by inserting thesequences encoding the proteins into a tetracycline-controlledexpression vector pUHD10-3. Control transfections employed vectorsencoding only the FAS-R and vectors encoding the FLAG-55.11 fusionprotein (the 55.11 protein being a p55-IC-specific binding protein ofwhich a portion containing amino acids 309-900 was fused (at itsN-terminal) to the FLAG octapeptide).

Following the transfection and incubation periods, the transfected cellswere treated with various concentrations of an anti-FAS-R monoclonalantibody (CH-11) which binds specifically to the extracellular domain ofFAS-R expressed by cells. This binding of anti-FAS-R antibody inducesthe aggregation of the FAS-R at the cell surface (much like the FAS-Rligand) and induces the intracellular signaling pathway mediated by theFAS-IC, resulting, ultimately, in cell death (FAS-R mediated cellcytotoxicity). The concentrations of the anti-FAS-R monoclonal antibody(CH-11) used were in the range of 0.01-10 μg/ml, usually concentrationssuch as 0.005; 0.05; 0.5 and 5 μg/ml. The cells were treated with theanti-FAS antibody in the presence of 10 μg/ml cycloheximide.

The results of the above analysis show that the expression of FAS-R inthe transfected cells conveys an increased sensitivity to the cytocidaleffects of the anti-FAS-R antibodies. Further, the co-expression of theregion in MORT-1 that contains the ‘death domain’ homology region andFAS-R strongly interferes with FAS-induced (i.e. FAS-R mediated) celldeath as would be expected from the ability of the MORT-1 ‘death domain’(DD) region to bind to the FAS-R ‘death domain’ (FAS-DD). Moreover,co-expression of the N-terminal part of MORT-1 and FAS-R does notinterfere with FAS-R-mediated cell death and, if at all, somewhatenhances the cytotoxicity (i.e., slightly increased cell death).

Thus, the above results clearly indicated that the MORT-1 protein hastwo distinct regions as far as binding to the FAS-IC and mediation ofthe cell-cytotoxic activity of the FAS-IC are concerned.

These results therefore also provide a basis for the use of differentparts (i.e., active fragments or analogs) of the MORT-1 protein fordifferent pharmaceutical applications. For example, the analogs orfragments or derivatives thereof of the MORT-1 protein which containessentially only the C-terminal portion of MORT-1 inclusive of its‘death domain’ region may be used for inhibiting FAS-R-mediatedcytotoxic effects in FAS-R containing cells or tissues and therebyprotect these cells or tissues from the deleterious effects of the FAS-Rligand in cases such as, for example, acute hepatitis. Alternatively,the analogs or fragments or derivatives thereof of the MORT-1 proteinwhich contain essentially only the N-terminal portion of MORT-1 may beused for enhancing the FAS-R-mediated cytotoxic effects in FAS-Rcontaining cells and tissues, thereby leading to the enhanceddestruction of these cells or tissues when desired in cases such as, forexample, tumor cells and autoreactive T and B cells. As detailed hereinabove, the above uses of the different regions of MORT-1 may be carriedout using the various recombinant viruses (e.g., Vaccinia) to insert theMORT-1 region-encoding sequence into specific cells or tissues it isdesired to treat.

Furthermore, it is also possible to prepare and use various othermolecules such as, antibodies, peptides and organic molecules which havesequences or molecular structures corresponding to the above notedMORT-1 regions in order to achieve the same desired effects mediated bythese MORT-1 regions.

Moreover, MORT-1 may be utilized to specifically identify, isolate andcharacterize other proteins which are capable of binding to MORT-1(i.e., MORT-1-binding proteins); see Reference Examples 2 and 3.

REFERENCE EXAMPLE 2 Isolation of a MORT-1 Binding Protein

(i) Two-hybrid Screen and Two-Hybrid β-Galactosidase Expression Test

In a manner analogous to the procedure described in Reference Example 1,using the intracellular domain of p55 TNF-R (p55 IC) and MORT-1 asbaits, and screening a human B-cell library, two cDNA clones wereobtained, which encode a protein product capable of binding to bothMORT-1 and p55-IC. Both clones were shown to have identical nucleotidesequences at the 5′ end (see co-owned, co-pending WO96/18641 andPCT/US96/10521).

(ii) Binding Properties of the Newly Cloned cDNA, in Two Hybrid Screens

Using the above-mentioned yeast two-hybrid procedure a constructcontaining the new MORT-1-binding protein cDNA was used as a “prey” towhich were added constructs of a number of “baits” in separatereactions, to determine the binding specificity of the MORT-1-bindingprotein encoded by this cDNA. These “baits” included constructs encodingMORT-1, portions of MORT-1 (MORT ‘head’, aal-117, MORT ‘tail’, aa130-245), the p55 IC (206-426 p55) or portion thereof (the ‘deathdomain’, 326-426 p55; and others upstream of the ‘death domain’ i.e.206-326). The results are shown below in Table 2. TABLE 2β-galactosidase Bait expression data MORT-1 +++ 130-245 MORT-1 + 1-117MORT-1 − 206-426 p55 +++ 326-426 p55 +++ 206-326 p55 − 206-308 p55 −206-345 p55 − p55 L35INI − Fas IC − 233-319 Fas − p75 IC − CD40 IC −pGBT10 − SNF1 − Cycline D − Lamin −

The above results of the two-hybrid β-galactosidase expression test ofthe binding of the clone to a large panel of baits confirmed that theprotein encoded by this clone binds specifically to the death domains ofboth the p55 TNF-R and MORT-1.

In general, the MORT-1 binding protein may be utilized directly tomodulate or mediate the MORT-1 associated effects on cells, or,indirectly, to modulate or mediate the FAS-R ligand effect on cells whenthis effect is modulated or mediated by MORT-1. The same holds true withrespect to other intracellular proteins or intracellular domains oftransmembrane proteins, as specifically demonstrated for the p55 TNF-Rherein.

MORT-1-binding proteins include those which bind specifically to theentire MORT-1 protein or those which bind to different regions of theMORT-1 protein, e.g., the above-noted N- and C-terminal regions ofMORT-1. The MORT-1-binding proteins which bind specifically to suchregions may be used to modulate the activity of these regions and hencethe specific activity of MORT-1 as determined by these regions.

REFERENCE EXAMPLE 3 Isolation and Characterization of the MACH Protein,Another MORT-1 Binding Protein

(i) Two-Hybrid Screen, Two-Hybrid β-Galactosidase Test, Sequencing andSequence Analysis

Using the procedure set forth in Reference Examples 1 and 2 above, afull length construct encoding human MORT-1 protein was employed as a“bait” in the yeast two-hybrid system to isolate a cDNA clone encodingan additional new MORT-1 binding protein. This new protein wasoriginally designated MORT-2, and now redesignated and referred to asMACH (for MORT-1 associated CED3 homolog), by virtue of itscharacteristics as detailed herein below.

This cDNA clone was sequenced by standard procedures as set forth inReference Examples 1 and 2 above. Sequence analysis by standardprocedures and computer programs (see Reference Examples 1 and 2)revealed that this cDNA has a novel sequence and encodes a novel protein(neither the DNA nor the amino acid sequences was found in GENBANK orPROTEIN BANK sequence databases). Further, the cDNA encoding MACHrevealed an ORF-B open reading frame which has strong homology to theregion preceeding (5′ upstream) the ‘death domain’ motif of the MORT-1protein (see Reference Example 1). In co-owned co-pending IsraelApplication Nos. 114615, 114986, 115319, 116588 and 117932 as well astheir corresponding PCT application No. PCT/US96/10521 there is shownthe structure of that part of the MACH cDNA clone which contains ORF-B(235 aa residues); the deduced amino acid sequence of the MACH ORF-B;and the nucleotide sequence of the MACH cDNA molecule. A region of ORF-Bshares high homology with the region of MORT-1 upstream of the MORT-1‘death domain” motif.

The yeast two-hybrid test was further applied to evaluate thespecificity of binding of MACH to MORT-1, in particular, to define theregion in MORT-1 to which MACH binds, as well as to determine which ofthe MACH ORFs interacts with MORT-1, the procedures being as set forthherein above in Reference Examples 1 and 2. Briefly, various MORT-1 andMACH constructs were prepared for testing the interaction of theproteins encoded by the Gal4 DNA-binding domain and activation domainconstructs within transfected SFY526 yeast cells as assessed by theB-galactosidase expression filter assay. The DNA-binding domainconstructs were prepared in pGBT9 vectors and the activation domainconstructs were prepared in pGAD-GM vectors. For the activation domainconstructs, the full-length MACH cDNA was used (MACH), as was aconstruct encoding only the ORF-B (MACH B) region. Control activationdomain constructs were those containing the full-length MORT-1 codingsequence (MORT 1, positive control) and those having no inserts, i.e.“empty” vectors (pGAD-GM). For the DNA-binding domain constructs, thefull-length MORT-1 cDNA was used (MORT 1), as were constructs encodingonly the MORT-1 upstream region (MORT-1DD aa 130-245). ControlDNA-binding domain constructs, which were constructed to determine alsothe specificity of the MACH binding, included constructs encoding lamin(Lamin), residues 287-461 of the intracellular domain of the human p75TNF-R (human p75 IC), cyclic D (cycD), SNF1, residues 206-426 of theintracellular domain of the human p55 TNF-R (human p55 IC), the ‘deathdomain’ region of the intracellular domain of the human Fas-R (human FasDD), residues 216-277 of the intracellular domain of the human CD40(human CD40 IC), vectors without insert or “empty” pGBT9 vectors (pGBT9,negative control), and a construct encoding the ORF-B region of MACH(MACH B). In the assay, the development of color was determined, wherethe greater the color development, the greater the interaction betweenthe constructs encoded by the DNA-binding domain and activation domain.Color development was depicted by symbols, where “+++” and “+” indicatethe development of a strong color within 30 and 90 min. of the assay,respectively, and “−−−” indicates the lack of development of colorwithin 24 hrs. of the assay. In cases where interactions were nottested, no symbol was indicated. The results of the various interactionsfor the above case are set forth in Table 3 below, and the results ofthe various interactions of the MACH isoforms are shown in abovementioned co-owned co-pending PCT/US96/10521 and its IL counterparts.TABLE 3 DOMAIN HYBRID MACH MACH B MORT 1 pGAD-GH DNA-Binding DomainHybrid MORT-1 +++ +++ +++ −−− Binding region in MORT-1 MORT1 (-117)MORT1DD (130-245) −−− −−− Specificity tests Lamin −−− −−− human p75 IC−−− cyc D SNF1 human p55 IC human FAS DD −−− human CD40 IC −−− pGBT9 −−−MACH B + + −−−

Thus, as arises from the results shown in Table 3 above, it is apparentthat:

-   -   (a) MACH binds to MORT-1 in a very strong and specific manner;    -   (b) The MACH binding site in MORT-1 occurs before (upstream of)        the ‘death domain’ motif in MORT-1, i.e., it is in the region of        MORT-1 defined by aa 1-117 of MORT-1;    -   (c) The ORF-B region of MACH is the MORT-1-interacting region of        the MACH protein; and    -   (d) The MACH ORF-B region is capable of self-association.        (ii) Cell-Cytotoxic Effects Mediated by the Self-Association        Capability of the MACH Protein

The observation that MACH can self-associate, in particular, that theORF-B region of MACH self-associates and the previous correlationbetween self-association and cell-cytotoxicity as observed for theintracellular domains of p55 TNF-R and FAS-R, and as observed for MORT-1(see Reference Example 1), suggested that MACH self-association may alsobe involved in cell-cytotoxicity.

In order to test this possibility, constructs encoding MACH wereprepared with a tetracycline-controlled expression vector (for detailssee Reference Example 1). These constructs were used to transfect HeLacells in which the vectors were transiently expressed. Besides the MACHconstructs, other control constructs were used to evaluate the effect oftransient expression on the viability of the HeLa cells to which theeffect of the MACH constructs could be compared. These other constructsincluded MORT-1, human FAS-IC and luciferase (Luc). In addition,co-transfection of the HeLa cells was also tested by using MORT-1 andMACH constructs to determine what effects the interaction between theseproteins would cause. After transfection the HeLa cells were incubatedand cell viability was evaluated 48 hrs. after transfection either inthe presence or the absence of tetracycline (1 μg/ml) to blockexpression. Cell viability was determined by the neutral red uptakeassay.

From the results of the above analysis, it was apparent that MACHinduces a dramatic cytotoxic effect in HeLa cells, i.e., the inducedoverexpression of MACH cDNA in HeLa cells, results in a dramaticcytotoxic effect. This cytotoxic effect is likely to be related to theself-association capability of MACH.

(iii) Northern Analysis

Using well-known procedures (see Reference Example 1), Northern analysisof several cell lines was carried out using the MACH cDNA as a probe.The results of this analysis show that in a large number of cell lines,in particular, CEM, Raji, Daudi, HeLa, Alexander, Jurkat and A673 celllines, there exist two hybridizing transcripts of approximately 3.2 kbin size.

In view of the above, the MACH protein, particularly the MACHβ1 protein(ORF-B of MACH) may be utilized directly to modulate or mediate theMORT-1 associated effects on cells, or, indirectly, to modulate ormediate the FAS-R ligand effect on cells when this effect is modulatedor mediated by MORT-1. The fact that MACH binds specifically to theupstream region of MORT-1 and shares homology with MORT-1 provides for aspecific way in which MACH or MACH ORF-B may be used to modulate thisspecific region of MORT-1 and hence the specific activity of MORT-1determined by this upstream region. Further, MACH or MACH ORF-B may beused as a modulator or mediator of intracellular effects in an analogousway to MORT-1 itself (see above) by virtue of MACH's ability toself-associate and induce cell-cytotoxicity on its own.

Further analyses of the MACH protein and the DNA sequences encoding ithave been performed as set forth herein below. Further, it was revealedthat ORF-B of MACH represents but one of a number of MACH isoforms.Hence, the MACH protein and the DNA sequences encoding it have now beenrenamed, as will become apparent from the following.

(a) Two Hybrid Screen for Proteins that Bind to MORT-1 Reveals a NovelProtein Which Shares a Sequence Motif with MORT-1:

As mentioned above, to identify proteins which participate in theinduction of cell death by MORT-1, the two-hybrid technique was used toscreen cDNA libraries for proteins that bind to MORT-1. A two-hybridscreen of a human B cell library (Dufee et al., 1993) using MORT-1 cDNAas bait yielded cDNA clones of MORT-1 itself, reflecting the ability ofthis protein to self-associate as well as clones of TRADD, to whichMORT-1 binds effectively (see Reference Example 2). The screen alsoyielded cDNA clones of a novel sequence whose product specifically boundto MORT-1. The protein, which initially was called MACH, and later,after finding that it occurs in multiple isoforms (see below), renamedMACHβ1, showed also an ability to bind in a two hybrid test to itself,yet was unable to bind to FAS-R (see above noted co-owned co-pendingPCT/US96/10521, which also includes all of the following analyses andresults obtained therefrom).

MORT-1 and MACHβ1 and their deletion constructs, as weil as MACHα1, aMACHα1 mutant in which the catalytic cysteine Cys₃₆₀ is replaced by Ser(MACHα1 (C360S)) and the intracellular domain of human FAS-R (Fas-IC),were expressed within transfected SFY526 yeast in Gal4 DNA bindingdomain and activation domain constructs (pGBT9 and pGAD-GH). Theirinteraction was assessed by a β-galactosidase expression filter assay asdescribed in Boldin et al., (1995b). The results are presented in termsof the time required for the development of strong color. None of theinserts examined interacted with a number of tested negative controls,including the intracellular domains of human p55 TNF receptor, p75 TNFreceptor and CD40, and lamin, cyclin D and ‘empty’ Ga14 vectors. MACHβ1was cloned by two hybrid screening of a Gal4 AD-tagged human B celllibrary (Durfee et al., 1993) for proteins that bind to MORT-1, usingthe HF7c yeast reporter strain. Except where otherwise indicated, allexperimental procedures for the findings presented are as describedabove (see also Boldin et al., 1995). Deletion analysis showed thatMACHβ1 binds to the N-terminal part of MORT-1, which is involved in celldeath induction (Chinnaiyan et al., 1995). MACHβ1 also self-associatedin the transfected yeast. However, it did not bind to several controlproteins and unlike MORT-1 was unable to bind to FAS-R. Expression ofMACHβ1 molecules in mammalian cells yielded a 34 kDa protein that boundto MORT-1 molecules co-expressed with it. It was also able to bind to aGST-MORT-1 fusion protein in vitro.

Comparison of the amino acid sequences in MACHβ1 and MORT-1 revealed ashared sequence motif (designated “Mort module”) in these two proteins,distinct from the death motif through which MORT-1 binds to FAS-R. Thismotif occurs once in MORT-1 and twice in MACHβ1. The same motif is foundalso in PEA-15, an astrocyte phosphoprotein of unknown function.Preliminary data suggest that the MORT motif is involved in the bindingof MACHβ1 (and of other MACH isoforms) to MORT-1.

The deduced amino acid sequence of MACHβ1 is presented in the abovenoted PCT/US96/10521 and its corresponding IL counterparts particularlyIL 117932. The two MORT modules are shown and the C-termini of the twoMACHβ1 deletion mutants employed are indicated. The sequence homology ofthe modules in MACHβ1, MORT-1 and the PEA-15 gene (accession numberX86809) was also presented in the above co-owned co-pendingapplications, in which identical and similar residues were denoted byboxed and shaded areas, respectively.

A diagrammatic representation of the death domain and MORT modules andof the CED3/ICE homology region in Fas/APO1, MACHβ1 and MACHα1, is alsopresented in the above co-owned applications.

The region in MORT-1 that contains this ‘MORT module’ has been shown totake part in cell death induction by this protein (see Reference Example1 above). It has been shown also to contribute to, though not to sufficein, the self association of MORT-1 (see Reference Example 1). Analysisof the binding properties of deletion constructs of MACHβ1 intransfected yeasts revealed similar involvement of the MORT modules inself-association of MACHβ1, as well as in its binding to MORT-1:Deletion constructs, in which the region below (downstream of) the MORTmodule was missing, were unable to bind to each other, yet maintainedthe ability to bind to the full length MORT-1 and to the full lengthMACHβ1. A further truncation in which part of the MORT module sequencewas also deleted, resulted in loss of the binding ability of theproteins. To further assess the involvement of the MORT modules in theseinteractions, deletion mutants of MACHβ1, fused with the FLAGoctapeptide (FLAG-MACHβ1), were expressed in HeLa cells and assessed fortheir binding in vitro to bacterial-producedglutathione-S-transferase-MORT-1 fusion protein (GST-MORT-1). Similarlyto the binding observed in the yeast two-hybrid test, this in vitrobinding was found to depend on interaction of the region within MACHβ1modules. ³⁵[S]-Metabolically labeled MACHβ1, MACHβ1 fused at itsN-terminus to the FLAG octapeptide (FLAG-MACHβ1), C-terminus truncationmutants of FLAG-MACHβ1, and, as a control, luciferase, were produced intransfected HeLa cells. Expression was done using atetracycline-controlled expression vector, in a HeLa cell clone (HtTA-1)that expresses a tetracycline-controlled transactivator.

Assessment of the expression of the proteins and their molecular sizeswas performed by immunoprecipitation from cell lysates, using anti-FLAGantibody. The antibodies used are as follows: Rabbit anti-MACHβ1 andanti-MORT1 antisera were raised against GST-MACHβ1 and GST-MORT1 fusionproteins. Mouse monoclonal antibodies against the FLAG octapeptide (M2)and against FAS/APO1 (CH11, Yonehara et al., 1989) were purchased fromEastman Kodak and Oncor (Gaithersburg, Md.) respectively. Mousemonoclonal anti-HA epitope antibody (12CA5, Field et al., 1988) andanti-TNF antibody were produced in our laboratory according to the usualmethods well known in the art. Results showing the affinity binding ofthe proteins to GST-MORT-1, adsorbed to glutathione-agarose beads (or,as a control, to GST or GST-fused to the intracellular domain ofFas-APO1); and the immuno-precipitations of the various MORT-1 and MACHfusion constructs using the various specific antibodies, are presentedin the above noted co-owned co-pending applications, in particular, inPCT/US96/10521 and IL 117932.

(b) MACH Occurs in Multiple Isoforms:

Northern analysis using MACHβ1 cDNA as a probe revealed low abundanttranscript(s) of approximately 3 kb in size in several different celllines. Briefly, Northern blot analysis of total RNA (14 μg/lane) or polyA⁺RNA (2 μg) from several cell lines, using MACHβ1 cDNA as probe wasperformed. The cell lines examined, T47D, CEM, Raji, Daudi, HeLa,Alexander, Jurkat and A673, are all of human origin and were derivedfrom a ductal carcinoma of the breast, an acute lymphoblastic T cellleukemia, a Burkitt lymphoma, a Burkitt lymphoma, an epitheloidcarcinoma, a human hepatoma, an acute T cell leukemia and arhabdomyosarcoma, respectively. The rather diffuse shape of thehybridizing band on Northern blots suggested that these transcripts areof heterogeneous sizes ranging between 2.85 and 3.5 Kb. Both the amountsand the sizes of the transcripts varied among different human tissuesand were not correlated with the expression of MORT1 or of FAS/APO1(Watanabe et al., 1992). In the testis and skeletal muscle, for example,MACH transcripts were barely detectable, even though these tissuesexpress significant amounts of MORT1. Conversely, resting peripheralblood mononuclear leukocytes, in which MORT1 expression is very low,were found to express MACH at high levels. Lectin activation of theleukocytes results in a marked change in the size pattern of MACHtranscripts, along with an induction of MORT-1.

Exploring the nature of this size heterogeneity, cDNA libraries werescreened for transcripts that hybridize with the MACHβ1 cDNA probe.MACHα1 and MACHα2 were cloned from a Charon BS cDNA library derived fromthe mRNA of human thymus. The library was screened under stringentconditions with a MACHβ1 cDNA probe, labeled using a random-priming kit(Boehringer Mannheim). The other MACH isoforms were cloned by RT-PCR,performed on total RNA from Raji (MACHα1, α2, β3, and β4) and Daudi(MACHα2, β2, β3, β4, and β5) human lymphoblastoid cells. Reversetranscriptase reaction was performed with an oligo-dT adapter primer(5′-GACTCGAGTCTAGAGTCGAC(T)₁₇-3′) (SEQ ID NO:12) and the SuperScript IIreverse transcriptase (GIBCO-BRL), used according to the manufacturer'sinstructions. The first round of PCR was performed with the Expand LongTemplate PCR System (Boehringer Mannheim) using the following sense andantisense primers: 5′-AAGTGAGCAGATCAGAATTGAG-3′ (SEQ ID NO:13),corresponding to nucleotides 530-551 of the MACHβ1 cDNA, and5′-GACTCGAGTCTAGAGTCGAC-3′ (SEQ ID NO:14), respectively. The secondround was performed with Vent polymerase (NEB) using the following senseand antisense nested primers: 5′GAGGATCCCCAAATGCAAACTGGATGATGAC-3′ (SEQID NO:15) and 5′-GCCACCAGCTAAAAACATTCTCAA-3′ (SEQ ID NO:16), derivedfrom the sequence of MACHβ1 cDNA, respectively. To confirm that MACHβ3and MACHβ4 have initiation codons, a more 5′ sequence of these isoformsfrom the RNA of Raji cells was cloned. The RT-PCR reaction, performedusing the oligo-dT adapter primer as described above, was followed bytwo rounds of PCR (with Vent polymerase (NEB)) using the following senseand antisense oligonucleotides: 5′-TTGGATCCAGATGGACTTCAGCAGAAATCTT-3′(SEQ ID NO:17) and 5′-ATTCTCAAACCCTGCATCCAAGTG-3′ (SEQ ID NO:18),derived from the sequence of MACHβ1. The latter oligonucleotide isspecific to the β-isoforms. Among the clones obtained in this way, thosefound to contain the nucleotides encoding for the amino acids of ‘block2’ (whose presence distinguishes MACHβ3 and MACHβ4 from MACHβ1 andMACHβ2) were fully sequenced. Nucleotide sequences in all clonedisoforms were determined in both directions by the dideoxy-chaintermination method. Only partial cDNA clones of MACHa3 and MACHβ2 wereobtained. This screening revealed the existence of multiple isoforms ofMACH MACH. The amino acid sequences of seven of these isoforms werestudied in detail. The results are illustrated diagrammatically andexemplified in the above co-owned co-pending applications, particularlyPCT/US96/10521 and IL 117932, where the amino acid sequences of three ofthe isoforms are compared with known homologs.

Lack of the 65 nucleotides which in MACHα1 encode for ‘block 2’ causesalteration in MACHβ1 and MACHβ2 of the reading frame of the nucleotidesthat encode for ‘block 3’. In those isoforms, therefore, thesenucleotides encode other amino acids which together constitute theirunique C-terminal region. On the other hand, in MACHβ3 and MACHβ4 thereading frame of block 3 is maintained, but absence of the nucleotidesthat encode the CED3/ICE region and part of the 3′ noncoding regionresults in alteration of the reading frame of nucleotides furtherdownstream. Because of this alteration, the most 5′ part of thisnoncoding downstream region does encode 10 amino acids, which constitutethe C-terminal region unique to these two isoforms.

The isoforms were cloned from a human B cell cDNA library (MACHβ1), froma human thymus cDNA library (MACHα1 and α2) and from the mRNA of thehuman lymphoblastoid cells Raji (MACH2α1, α2, α3, β3 β4, and β5) andDaudi (MACHα2, β2, β3, β4 and β5). Cloning from the mRNA of the Raji andDaudi cells was done by RT-PCR, using oligonucleotides corresponding toa 3′ noncoding region and to a sequence within the second MORT module inMACHβ1. The starting codon of clones isolated in that way is thereforelocated within the second MORT module.

The sequences in the different isoforms relate to each other as follows:(a) All the MACH isoforms share a common 182-amino acid N-terminalregion which encompasses the MORT modules, yet vary carboxy terminally(3′ downstream) to these modules, as well as in their noncoding regions.(b) On the basis of their C terminal sequences, the isoforms fall intotwo subgroups: four isoforms defined as subgroup β, have differentC-termini due to alteration in the reading frame. Two (MACHβ1 AND β2)share the C-terminus found in the isoform initially cloned in thetwo-hybrid screen and two (MACHβ3 and β4) share a different C-terminus;three isoforms, defined as subgroup α, have a much longer C-terminalregion that closely resemble proteases of the CED3/ICE family (seebelow); (c) The regions extending between the MORT module region and theC terminal region that defines the subgroups varied from one isoform toanother. However, close examination showed that these intermediateregions consist of different combinations of the same three amino acidsequence blocks (blocks 1, 2 and 3). The variations of amino acidsequence among the different clones reflect two kinds of variations innucleotide sequence, that most likely occur by alternative splicing: (a)insertion or absence of either of two nucleotide sequences, one of 45nucleotides (nts) and the other of 65 nts, or of both, below thenucleotides encoding Lys184; (b) presence of an additional insert withinthe region which in MACHβ1 constitutes the 3′ noncoding part. Thesevariations affect both the reading frame and the length of the protein.

Part of the MACH isoforms encompass a CED3/ICE homolog. Data banksearch. revealed that the C terminal region of MACHα isoforms includingblock 3 and the sequence extending downstream of it, closely resembleproteases of the CED3/ICE family. A sequence comparison of this regionin MACH and the various known human members of this family as well asthe Caenorhabditis elegans ced3 protein was performed (Ellis andHorvitz, 1986; Yuan et al., 1993), and the known human proteases of theCED3/ICE protease family: CPP32 (Fernandes-Alnemri et al., 1994), alsocalled apopain (Nicholson et al., 1995) and Yama (Tewari et al., 1995b),Mch2α (Fernandes-Alnemri et al., 1995), Ich-1 (Wang et al., 1994; thehuman homolog of the mouse Nedd2 protein, Kumar et al., 1994),ICE_(rel)II (<umday et al., 1995), ICE_(rel)II (Munday et al., 1995),also called TX and Ich2 (Faucheu et al., 1995; Kamens et al., 1995), andICE (Thornberry et al., 1992; Cerretti et al., 1992).

The above C-terminal region of MACH most closely resembles CPP32 (with41% identity and 62% homology) and CED3 (with 34% identity and 56%homology). It shows a significantly lesser similarity to ICE (with 28%identity and 50% homology) and to its closely related homologsICE_(rel)II (also called TX and Ich2) and ICE_(rel)III. The similaritywas observed throughout almost the whole region starting from Tyr226within block 3, to the C terminus of the MACHα isoforms.

Two points of similarity are particularly notable:

-   -   (a) All known proteases of the CED3/ICE family cleave proteins        at sites defined by the occurrence of Asp at the P1 position and        a small hydrophobic amino acid residue at P1′. Their specificity        differs, though, with regard to other structural features of the        substrate, including the nature of the residues at positions        P2-P4. Accordingly, the active site residues involved in        catalysis (corresponding to His237, Gly238 and Cys285 in ICE)        and in the binding pocket for the carboxylate side chain of the        P1 Asp (Arg179, Gln283, Arg341 and probably also Ser347) are        conserved among these proteases. These residues are also        conserved in MACHα1. There is one exception, though—a        conservative change of Ser to Thr at the site corresponding to        Ser347 of ICE. Another slight, yet potentially important,        sequence difference between MACHα isoforms and other members of        the protease family is an Arg to Gln replacement of the residue        corresponding to Arg286 of ICE. This residue, which is adjacent        to the putative catalytic cysteine residue, is fully conserved        in all other CED3/ICE family members. Also part of the residues        at the sites located close to the substrate P2-P4 residues        differ in the MACHα isoforms from those found in other CED3/ICE        family members.

(b) Proteases of the CED3/ICE family contain sites of autocleavage.Several of the proteases are known indeed to be self-processed, and todepend on this processing for displaying maximal catalytic activity.Their fully bioactive form is composed of two noncovalently-associatedcleavage products, which differ in size (p20 and p17 in ICE; p17 and p12in CPP32). Presence of potential sites of autocleavage in other membersof the family suggests that they are subject to similar processing, and,similarly, depend on this processing for exhibiting maximal activity.Such potential sites of autocleavage occur in MACHα1 almost at the samelocations as in the CPP32. The site corresponding to the N terminus ofthe p17 subunit of CPP32 is located in the second conserved block ofamino acids, just a few amino acids upstream to the N terminus of theCED3/ICE-homology region (below Asp216). The site corresponding to thepoint of cleavage between the two subunits of CPP32 is located, as inall other members of the CED3/ICE family that are known to be cleaved, afew amino acids downstream to the catalytic cysteine residue (belowAsp374). This conservation suggests that the CED3/ICE homology region inMACHα1 is subject to proteolytic processing. The sizes of the twoexpected products of this cleavage are very close to that of the twosubunits of the processed CPP32 molecule.

(c) The CED3/ICE Homology Region in MACH has Proteolytic Activity.

To find out if the CED3/ICE homology region in MACHα possessesproteolytic activity, applicants expressed the region that extends fromthe potential cleavage site upstream to this region, between Asp216 andSer217, till the C terminus of the protein in bacteria, as a GST fusionprotein. The bacterial lysates were examined for ability to cleavefluorogenic peptide substrates, shown before to be cleaved by otherCED3/ICE homologs. Two substrate peptides were used: The first,Acetyl-Asp-Glu-Val-Asp-a-(4-Methyl-Coumaryl-7-Amide) (AC-DEVD-AMC) (SEQID NO:10), corresponds to a sequence in poly (ADP-ribose) polymerase(PARP), a nuclear protein found to be cleaved in cells shortly afterFAS-R stimulation (Tewari et al., 1995b), as well as in other apoptopicprocesses (Kaufmann, 1989; Kaufmann et al., 1993; Lazebnik et al.,1994). This fluorogenic substrate is cleaved effectively by CPP32. Thesecond fluorogenic substrate, Acetyl-Tyr-Val-Ala-Asp-AMC (Ac-YVAD-AMC)(SEQ ID NO:11), corresponds to a substrate site for ICE in the IL-1βprecursor. This fluorogenic substrate is cleaved by ICE. Lysates ofbacteria expressing the CED3/ICE homology region in MACHα1 cleavedeffectively the PARP sequence-derived fluorogenic substrate. They had nomeasurable proteolytic activity, though, against the IL-1β-precursorsequence-derived fluorogenic substrate (controls), Ac-YVAD-AMC, which isan ICE cleavage site in IL-1β precursor (Thornberry et al, 1992). Theproteolytic activity was blocked by iodacetic acid (5 mM), confirmingthat it is mediated by a thiol protease. No cleavage was observed withlysates containing the GST-fused MACH CED3/ICE-homology region in whichthe catalytic cysteine residue Cys₃₆₀ was replaced by Ser. Also, lysatesfrom bacteria that expressed the full-length MACHα1 protein as aGST-fusion protein did not cleave Ac-DEVD-AMC, probably because of theabsence of bacterial enzymes capable of processing the full-lengthmolecule. Nor did cleavage occur with lysates containing either of thetwo potential cleavage products of the CED3/ICE homology region.

The kinetics of cleavage of the PARP sequence-derived fluorogenicsubstrate, Ac-DEVD-AMC (50 μM), by extracts of E. coli expressing aGST-fusion protein of the CED3/ICE homology region in MACHα1 (Ser217through the C-terminus of the protein) was shown as compared to the lackof cleavage by extracts of bacteria expressing GST-fusion proteins ofthe full-length MACHα1 molecule or of either one of the two potentialproteolytic products of the CED3/ICE homology region (Ser217 till Asp374and Asp374 through the C-terminus of the protein).

Further, the substrate concentration-dependence of the cleavage ofAc-DEVD-AMC, incubated for 180 min. with extracts of bacteria expressingthe MACHα1 CED3/ICE homology-region in fusion with GST was shown. Nocleavage was observed in the presence of iodoacetic acid (5 mM). Theextracts had no activity on Ac-YVAD-AMC, a fluorogenic substratecorresponding to a substrate site for ICE in the IL-1β precursor.

Briefly, the GST-fusion proteins were produced in XL1-blue bacteriausing the pGEX3 expression vector. The bacteria were lysed by sonicationin a buffer containing 25 mM HEPES (pH 7.5), 0.1%3-[3-cholamidopropyl)dimethylamino]-1-propanesulfonate, 5 mM EDTA and 2mM DDT, followed by centrifugation at 16,000× g for 10 min. SDS-PAGEanalysis confirmed the presence of similar levels of the various fusionproteins in the lysates. 50 μl aliquots of the extracts (4 mg/ml oftotal protein) were incubated at room temperature for the indicatedperiods in a 500 μl total volume reaction with the fluorogenicsubstrates, at the indicated concentrations. AMC release was measured byspectro-fluorometry at an excitation wavelength of 380 nm and anemission wavelength of 460 nm. The concentration of AMC was determinedfrom a standard curve. Both fluorogenic substrate peptides were obtainedfrom Peptide Institute Inc. (Osaka, Japan). Other CED3/ICE proteaseswere shown to exhibit full activity only after proteolytic processing,which occurs either by self-cleavage, or via their cleavage by otherproteases (reviewed in Kumar, 1995; Henkart, 1996). Applicants'observation that lysates of bacteria that express GST-MACHα1 moleculesdo not possess enzymatic activity, as opposed to the activity observedin lysates of bacteria that express the CED3/ICE homology region,suggests that processing is also required for MACHα activity. The way inwhich MACHα processing occurs within the mammalian cell, and how thisprocessing is brought about by FAS-R or p55-R triggering, is not known.MORT-1 has been shown to bind in cells to activated FAS-R together withsome other proteins (Kischkel et al., 1995). These proteins are likelyto include MACHα1 and other MACH isoforms. It seems plausible that thebinding of MORT-1 in association with MACHα to FAS-R brings togetherseveral MACH molecules, or induces conformational changes in them, andthat these changes either trigger autolytic processing of MACHα or makeMACHα susceptible to cleavage by other proteases. Stimulation of p55-Rmay trigger self-processing of MACHα in a similar, though less directmanner, by bringing together several TRADD molecules, or inducing aconformational change in them, which in turn induces a change in theformation or state or aggregation of MORT-1 and its associated MACHmolecule.

The substrate specificity of MACHα seems to be rather ‘death oriented’.Although it could cleave a substrate peptide corresponding to a cleavagesite in the death substrate PARP (Ac-DEVD-AMC), MACHα showed noproteolytic activity towards a peptide corresponding to the site ofprocessing of the IL-1β precursor by ICE (Ac-YVAD-AMC). Identificationof the cellular proteins that serve as substrates for cleavage: by MACHαwill elucidate the more downstream events in death induction by thisprotease. Likely substrates for MACHα cleavage are other members of theCED3/ICE family, like CPP32 and ICE. Some of these proteases are indeedprocessed after FAS-R or TNF receptor-triggering (Miura et al., 1995;Schlegel et al., 1996; Chinnaiyan et al., 1996). Perhaps proteases thatdo not belong to the CED3/ICE family are also activated by MACHα, eitherdirectly or through the action of other CED3/ICE proteases. Involvementof multiple proteases in the cell death process is consistent with thereported ability of inhibitors of various proteases, includinginhibitors of serine proteases and an inhibitor of ICE cleavage as wellas antisense ICE cDNA, to protect cells from FAS-R and TNFreceptor-induced toxicity (Weitzen and Granger, 1980; Ruggiero et al.,1987; Enari et al., 1995; Los et al., 1995).

A variety of other enzymes, including phospholipases, sphingomyelinasesand protein kinases, may participate in cell death induction by the TNFreceptors and FAS-R (see Eischen et al., 1994; Vandenabeele et al.,1995; Cifone et al., 1995 and references therein). Some of these enzymesmay become activated by the proteolytic cleavage initiated by MACHα. Italso seems possible, however, that at least part of these otherdeath-related activities are stimulated by distinct signaling routes,independently of MACHα stimulation. Involvement of more than onesignaling cascade in the induction of cell death, some common to p55-Rand FAS/APO1 and some induced by only one of them, would be consistentwith report on both shared and distinct features of cell death processesinduced by the two receptors (Grell et al., 1994; Schulze-Osthoff etal., 1994; Wong and Goeddel, 1994; Clement and Stamenkovic, 1994).

(d) MACHα1 Binds to MORT1 as Well as to MACHβ1:

To find out if MACHα1 can bind to MORT1, as does MACHβ1, the interactionof the proteins within transfected yeasts was first examined. MACHα1appeared to have a significant cytotoxic effect on the yeasts. Thiseffect was manifested in a marked decrease in the yield of colonies inyeasts that expressed the protein in the activation domain (AD) vector(whose expression level is higher than that of the DNA binding domain(DBD) vector). On the other hand, MACHβ1 in which the catalytic cysteineresidue, Cys₃₆₀, was replaced with Ser (MACHα1 (C360S)) was notcytotoxic to either mammalian cells (see below), or yeast. Like MACHβ1,MACHα1 (C360S) bound in. transfected yeast to MORT-1 and also to itself.It also bound to MACHβ1. Also, yeast expressing the wild-type MACHα1together with MORT-1 or MACHβ1 exhibited interaction of the transfectedproteins. The intensity of the lacZ-product color varied, however, amongthe yeast colonies; in yeasts transfected with MACHα1 in both the AD andthe DBD vectors no color product was observed, probably because of thecytotoxic effect of the wild-type MACHα1. Yet, in spite of thisvariation, yeasts expressing MACHα1 either in combination with MORT1 orin combination with MACHβ1 scored clearly positive for interaction ofthe transfected proteins. Unlike MACHβ1, MACHα1 did not exhibitself-interaction in the two hybrid test.

Both MACHα1 (C360S) and MACHβ1 coimmunoprecipitated with MORT-1 fromlysates of human embryonic kidney 293-EBNA cells, indicating that theybind to MORT-1 also in mammalian cells. Testing further if MACHα1 canbind to MORT1 also within mammalian cells, MACHα1 or MACHβ1, fused withthe FLAG octapeptide was expressed, together with HA epitope-taggedMORT1 molecules. ³⁵[S] metabolically labeled MACHα1 and MACHβ1 fused attheir N-termini to the FLAG octapeptide (FLAG-MACHα1 and β1), and MORT1fused at its N terminus to the HA epitope (Field et al., 1988) wereexpressed in HeLa cells. Immunoprecipitation of the proteins fromlysates of the cells was performed using mouse monoclonal antibodiesagainst the FLAG octapeptide (M2; Eastman Kodak), HA epitope (12CA5,Field et al., 1988) or the p75 TNF receptor (#9, Bigda et al., 1994) asa control. The proteins were analyzed by SDS-polyacrylamide gelelectrophoresis (12% acrylamide), followed by autoradiography. BothMACHα1 and MACHβ1 co-immunoprecipitated with MORT1 from lysates of thecells, indicating that they bind to MORT1. The effectivity ofinteraction of MACHα1 with MORT1 appeared to be lower than that ofMACHβ1.

(e) MACH Molecules that Contain the CED3/ICE Homology Region can MediateCell Death:

To explore the involvement of MACH in cell-death induction, the effectof overexpression of various MACH isoforms on cell viability wasexamined. The test was performed by transfecting MACH expression vectorstogether with a β-galactosidase. expression vector as a transfectionmarker into human embryonic kidney 293-EBNA cells and breast carcinomaMCF7 cells.

In brief, 293-EBNA cells, MCF7 human breast carcinoma cells and HeLaHtTA-1 cells were grown in Dulbecco's modified Eagle's minimal essentialmedium supplemented with 10% fetal calf serum, nonessential amino acids,100 U/ml penicillin and 100 μg/ml streptomycin. Cell tissue culturedishes (5×10⁵ 293-EBNA cells, 3×10⁵ MCF7 cells or 3×10⁵ HeLa cells in6-cm dishes) were transiently transfected, using the calcium phosphateprecipitation method, with the cDNAs of the indicated proteins togetherwith the β-galactosidase expression vector. In one set of experiments,each dish was transfected with 3.5 μg of the MACH construct and 1.5 μgof pSV-β-gal; and in another set of experiments, each dish wastransfected with 2.5 μg of the indicated MACH or MORT1 construct (or, ascontrol, empty vector) and 1.5 μg of pSV-β-gal. The cells were rinsed 6to 10 h after transfection. The 293-EBNA and MCF7 cells were incubatedfor a further 18 h without additional treatment. The HeLa cells wereincubated for 26 h after transfection and then for 5 h in the presenceof either anti-Fas.APO1 antibody (CH11, 0.5 μg/ml) or TNF (100 ng/ml),together with cycloheximide (10 μg/ml). The extent of cell death at theend of the incubation periods was assessed by determination ofβ-galactosidase expression, as described by Kumar et al., 1994.

Cultures transfected with an expression vector of either MACHα1 orMACHα2 exhibited massive cell death, manifested by cell rounding,blebbing, contraction, and finally detachment of cells from the dish. By20 h after transfection, the majority of the transfected cells,identified by β-galactosidase staining (X-Gal), showed condensedmorphology typical of apoptosis. In contrast, cells expressing the emptyvector remained viable.

To examine the involvement of the CED3/ICE homology region within theMACHα isoforms in their apoptopic effects, cells were transfected withthe expression vector for the MACHβ1 isoform, which lacks the CED3/ICEhomology region, as well as with expression vectors for MACHα3, whichlacks an N-terminal part of the region, and with expression vectors forMACHα1(C360S) and for a C-terminally truncated mutant of MACHα1(MACHa1(1-415)), which lacks one of the residues believed to be criticalfor CED3/ICE protease function (corresponding to Ser₃₄₇ in ICE). Nodeath (beyond the slight amount observed in cells transfected with anempty expression vector) occurred in 293-EBNA or MCF7 cells transfectedwith the expression vectors for MACHα3, MACHα1(1-415) or MACHα1(C360S).Moreover, cells transfected with MACHα1 together with these vectors alsoexhibited very little cell death, indicating that MACH molecules thatcontain an incomplete CED3/ICE region have a negative dominant effect onthe activity of the wild-type molecules. Cultures expressing MACHβ1,which does not contain the CED3/ICE region at all, did exhibit someslight cell death. This effect of MACHβ1, which most probably resultsfrom activation of endogenous MACHα1 molecules, was for some reason morepronounced in transfected HeLa cells. Moreover, in HeLa cells MACHα3,MACHα1(1-415) and MACHα1(C360S) were also somewhat cytotoxic.

MACHα activity appears to constitute the most upstream enzymatic step inthe cascade of signaling for the cytocidal effects of FAS/APO1 andp55-R. The ability of MACHβ1 to bind to both MORT-1 and MACHα1 suggeststhat this isoform enhances the activity of the enzymatically activeisoforms. It is possible that some of the MACH isoforms serve additionalfunctions. The ability of MACHβ1 to bind to both MORT-1 and MACHα1suggests that this isoform might enhance the activity of theenzymatically active isoforms. The mild cytotoxicity observed in293-EBNA and MCF7 cultures transfected with this isoform and the rathersignificant cytotoxic effect that it exerts in HeLa cells probablyreflect activation of endogenously expressed MACHα molecules uponbinding to the transfected MACHβ1 molecules. Conceivably, some of theMACH isoforms could also act as docking sites for molecules that areinvolved in other, non-cytotoxic effects of FAS/APO1 and TNF receptors.

(f) Blocking of MACHα Function Interferes with Cell Death Induction byFas/APO1 and P55-R

To assess the contribution of MACHα to Fas/APO1 and p55-R cytotoxicity,MACHα3, as well as the nonfunctional MACHα1 mutants, MACHα1(1-415) andMACHα(C360S), were expressed in cells that were induced to exhibit thiscytotoxicity. p55-R-induced cytotoxicity was triggered in the 293-EBNAcells by transient over-expression of this receptor (Boldin et al.,1995a), and Fas/APO1 cytotoxicity by over-expression of chimericmolecules comprised of the extracellular domain of the p55-R and thetransmembrane and intracellular domains of Fas/APO1. This chimera had afar greater cytotoxic effect than that of the normal Fas/APO1. Cytotoxicactivities in HeLa cells was also induced by treating them with TNF oranti-Fas/APO1 antibody in the presence of the protein-synthesis blockercycloheximide. The HeLa cells were made responsive to Fas/APO1 bytransient expression of this receptor. In all systems examined, MACHα3and the nonfunctional MACHα1 mutants provided effective protectionagainst the cytotoxicity induced by Fas/APO1 or p55-R triggering. Suchprotection was also observed, as previously reported (Hsu et al., 1996;Chinnaiyan et al., 1996), in cells transfected with a MORT-1 N-terminaldeletion mutant that lacks the MACH-binding region (MORT1(92-208)).These protective effects indicate that MACHα is a necessary component ofboth the Fas/APO1- and the p55-R-induced signaling cascades for celldeath.

MACH is expressed in different tissues at markedly different levels andapparently also with different isotype patterns. These differencesprobably contribute to the tissue-specific features of response to theFAS/APO1 ligand and TNF. As in the case of other CED3/ICE homologs (Wanget al., 1994; Alnemri et al., 1995), MACH isoforms containing incompleteCED3/ICE regions (e.g. MACHα3) are found to inhibit the activities ofcoexpressed MACHα1 or MACHα2 molecules; they are also found to blockdeath induction by FAS/APO1 and p55-R. Expression of such inhibitoryisoforms in cells may constitute a mechanism of cellular self-protectionagainst FAS/APO1- and TNF-mediated cytotoxicity. The wide heterogeneityof MACH isoforms, which greatly exceeds that observed for any of theother proteases of the CED3/ICE family, should allow a particularly finetuning of the function of the active MACH isoforms.

EXAMPLE 1 Cloning and Isolation of the G1 Protein Which Binds to theMORT-1-Binding Protein Mch4

(i) Two-Hybrid Screen and Two-Hybrid β-Galactosidase Expression Test

Using the procedures set forth hereinabove in the Reference Examples1-3, a new protein designated G1 was isolated which is apparentlyhomologous to and hence possibly a member of the family of ICE-likeproteases. The G1 protein contains two modules with homology to the MORTmodules, namely with homology to the, MORT-1 N-terminal part, the MORTmodules of the MACH proteins (see above reference Example 1-3 and Boldinet al., 1996) and the MORT modules of another related MORT-1-bindingprotein Mch4 (see Fernandes-Alnemri et al., 1996; Srinivasula et al.,1996). Further, the G1 protein has an enzymatic (protease-like) regionthat is homologous to the enzymatic (protease) regions of proteases ofthe CED3/ICE family (for example, the protease regions of the MACHproteins and Mch4, and others).

Briefly, a clone of protein G1 (‘clone GI’) was obtained followingtwo-hybrid screening of a human Jurkat T cell cDNA library using theprotein Mch4 as ‘bait’. A Gal4 activation domain-tagged human Jurkat Tcell cDNA library was used, and screening was performed using the HF7cyeast reporter strain (Clontech, Palo Alto, Calif.) in the absence of3-aminotriazole according to the Matchmaker™ Two-Hybrid System Protocol(Clontech). The Mch4 sequence was obtained from the EMBL database. Usingthis obtained sequence, PCR-primers were designed by OLIGO4™ softwareand the DNA fragment corresponding to the coding part of Mch4 wasobtained by reverse transcriptase-PCR (RT-PCR) from the total RNAobtained (by standard methods) from primary Human Umbilical VeinEndothelial cells. This coding part of Mch4 was then cloned into thepGADGH vector (Clontech) and used as a bait, as noted above, in thetwo-hybrid screening procedure. In this two-hybrid screen 11 clones wereobtained, all coding for a protein which was apparently a splice variantof the protein containing two motifs of homology to MORT-1. Analysis ofthe preliminary partial sequence of G1 and sequences in the ‘dbest’database and Human Genome Database level 1 enabled the obtention of anumber of expressed sequence tags (est) containing parts of the sequenceof the clone G1. Sequence analysis of these est revealed that there arepossibly a number of splice variants of protein G1 that contain asequence stretch coding for a protein motif that is homologous toICE-like proteases and that this sequence stretch is located 3′ to theG1 sequence obtained in the two-hybrid screening.

To obtain a full sequence of the isoform of G1 which contains theprotease-like enzymatic region, reverse transcriptase reaction wasperformed on total RNA obtained from various cell lines using as aprimer an oligonucleotide containing a 15dT stretch and an adaptorsequence to yield cDNA molecules. These cDNA molecules were then used astemplates in a PCR reation in which the PCR primers were designed andsynthesized in the form of oligonucleotides having a sequence obtainedfrom the 5′ non-coding part of G1 and an adaptor sequence, these primersbeing used in a first-round PCR reation. Subsequently in thesecond-round of the above PCR reaction additional oligonucleotideprimers were used having a sequence from the 5′ coding part of G1inclusive of the initiator ATG, as well as an adaptor sequence. In thisway it was possible to obtain the full sequence of an apparent splicevariant of G1 protein which contains the enzymatic (protease-like)region. This represents but one of the suspected G1 isoforms.

A preliminary sequence of one such G1 isoform, a G1 splice variant, isdepicted in FIG. 1A+B, in which FIG. 1A is the nucleotide sequence andFIG. 1B is the deduced amino acid sequence of an ORF starting from ATG(nucleotide No. 482) and terminating at TAA (nucleotide 1921), thesestart and terminator nucleotides being indicated by asterises (*) in thenucleotide sequence. The G1 splice variant of FIG. 1 has also beenputatively designated ‘G1α’.

In FIG. 2 there is schematically depicted the preliminary sequence ofanother G1 isoform, a short G1 splice variant having two MORT MODULES,putatively designated ‘G1β’, in which the upper sequence is thenucleotide sequence and the lower sequence is the deduced amino acidsequence of an ORF starting at nucleotide No. 482 (ATG) and terminatingat nucleotide No. 1145 (TGA), the start and terminator codons beingindicated by asterises (*) in the nucleotide sequence.

Furthermore, it should be noted that the originally isolated G1 cloneobtained using the Mch4 sequence as ‘bait’ was at least part of theshort splice variant of G1, called G1β (see FIG. 2). This originallyisolated G1 clone was a partial clone of a novel cDNA, which like MACH(CASP-8) and Mch4 (CASP-10), contained two ‘death domain motifs/MortModules (or ‘death effector domains’—DED) just downstream of itsN-terminus (see also FIG. 3). Using this original clone it was thenpossible to isolate and characterize the cDNA clones for the largersplice variant G1α and the shorter splice variant G1β. The larger splicevariant had a C-terminal region with homology to the protease region ofcaspases and hence is likely to be a new member of the caspase family.As such G1 has also been designated CASH for ‘caspase homolog’. Acomparison of the G1α (CASHα) and G1β (CASHβ) sequences with othercaspase sequences was carried out (for further details, especially theisolation of the mouse G1 sequence, see below and above under ‘BriefDescription of the Drawings’ with respect to FIG. 3). The results ofthis comparison are set forth schematically in FIG. 3, the full detailsof which, in particular, the key to the indicated sequences in thisfigure are noted above under ‘Brief Description of the Drawings’.

Following the above initial cloning and sequencing of G1, in particularthe G1α and G1β isoforms, further analysis of these proteins wasperformed:

(ii) Northern Analysis and Additional Sequence Analysis:

Northern blot analysis revealed that the G1 protein exists in at leastthree distinct transcript sizes, 2, 2.4 and 4.4 kb, whose proportionsvary greatly among different tissues (FIG. 4). To obtain the full-lengthcDNA of G1 (CASH), a human skin fibroblast cDNA library (Clontech) wasscreened with a cDNA probe corresponding to the G1 sequence. Two cDNAspecies, apparently corresponding to two splice variants of G1 wereobtained (see also above). The proteins encoded by these two cDNAsshared the death-effector domain-containing N-terminal region, but theirC-termini differed. One (G1β=CASHβ) had a short C-terminus,corresponding to that of the originally cloned cDNA. The other(G1α=CASHα) had a long C terminus.

The amino acid sequence in this longer C-terminal region of G1α showedrather high homology to those of the protease-precursor regions in MACH(CASP-8) and Mch4 (CASP-10) (see also FIG. 3). However, G1α lackedseveral of the residues believed to be crucial for protease activity,suggesting that the protein may be devoid of cysteine protease activity.Interestingly, G1 a contains a caspase-substrate sequence at the sitecorresponding to the proteolytic-processing site within the proteaseregions in MACH (CASP-8) and Mch4 (CASP-10) (shaded in FIG. 3).Preliminary data suggest that G1α can indeed be cleaved at this site byMACH (data not shown).

Based on the nucleotide sequence of an EST clone found to correspond tothe mouse homologue of part of the ‘death domain’ (DED) region in G1,the cDNAs of both the mouse CASHα and CASHβ splice variants were clonedfrom mouse liver mRNA by RT-PCR. An EST clone (GenBank accession no.AA198928) was identified as the mouse homologue of part of the DEDregion in G1. Based on this sequence the mouse G1α (CASHα) and G1β(CASHβ) splice variants from mouse liver mRNA were cloned by RT-PCR. Thereverse transcriptase reaction was performed with an oligo-dT adapterprimer (5′-GACTCGAGTCTAGAGTCGAC(T)₁₇-3′) (SEQ ID NO:12) and the AMVreverse transcriptase (Promega), used according to the manufacturer'sinstructions. The first round of PCR was carried out with the ExpandLong Template PCR System (Boehringer Mannheim) using the following senseand antisense primers: 5′-GGCTTCTCGTGGTTCCCAGAGC-3′ (SEQ ID NO:19), and5′-GACTCGAGTCTAGAGTCGAC-3′ (base pairs 1-20 of SEQ ID NO:12) (adapter)respectively. The second round was performed with Vent polymerase (NEB)using the nested sense primer: 5′-TGCTCTTCCTGTGTAGAGATG-3′ (SEQ IDNO:20), and adapter.

Sequence comparison revealed high conservation throughout the G1α(CASHα) molecule (71% identity in DED region and 59% in proteasehomology region), suggesting that both the DED and protease-homologyregions in the protein contribute to its function (see FIG. 3).

(iii) Two-Hybrid Analysis for Binding Specificity of G1 Isoforms:

Two-hybrid testing of the interactive properties of G1α (CASHα) and G1β(CASHβ) (FIG. 5) revealed that both variants interact with MORT1/FADDand MACH (CASP-8), most probably through their shared DED regions.Notably, although initially cloned by two-hybrid screening for proteinsthat bind to Mch4 (CASP-10), G1β (CASHβ) was found in this test to bindweakly to Mch4 (CASP-10) and G1α (CASHα) appear to bind only weakly toMch4. It should however be noted that the initial two-hybrid screen toclone the G1 proteins differed from this two-hybrid screen to assaybinding specificity and hence, in reality it would appear that G1α andG1β both bind to MACH and Mch4 as the original cloning results show. Thetwo G1 variants also self-associated and bound to each other, but didnot bind RIP or TRADD (adapter proteins which, like MORT1/FADD, containdeath domains but lack DEDs), nor did they bind to a number ofirrelevant proteins (e.g. lamin) used as specificity controls.

To examine further the function of G1, its two variants were expressedtransiently in HeLa and 293-T cells and there was assessed the effectsof the transfected proteins on the p55-R (CD120a)-induced signaling forcytotoxicity triggered by TNF, or by overexpression of the receptor aswell as on the FAS-R (CD95)-induced signaling for cytotoxicity triggeredby antibody cross-linking of FAS-R, or by overexpression of a chimericreceptor comprised of the extracellular domain of p55-R (CD120a) and theintracellular domain of FAS-R (CD95) (see FIG. 6). In both cell lines,expression of G1β (CASHβ) by itself had no effect on cell viability, butit strongly inhibited the induction of cell death by p55-R (CD120a) aswell as by FAS-R (CD95). Expression of the G1α (CASHα) variant affectedthe two cell lines very differently. In HeLa cells it inhibited thecytotoxicity of p55-R (CD120a) and FAS-R (CD95), similarly to G1β(CASHβ). In the 293-T cells, however, it resulted in markedcytotoxicity. Similar cytotoxicity was observed when the G1α protein wasexpressed in 293-EBNA cells (not shown). This cytotoxic effect could becompletely blocked by coexpression of p35, a baculovirus-derived caspaseinhibitor (for p35 see also Clem et al., 1991; Xue and Horvitz, 1995).

To assess the contribution of the region of protease homology in G1α(CASHα) to its cytocidal effect, the functions of two mutants of theprotein were examined. These mutants were: G1/CASHα(1-385) andG1/CASHα(1-408), with C-terminal deletions at the region correspondingto that part of the protease domain from which the small subunit of themature protease is derived. Both mutants were devoid of any cytotoxiceffect. Moreover, like G1β (CASHβ) they protected the 293 cells fromdeath induction by p55-R (CD120a) and FAS-R (cd95) (FIG. 6C and D).

It should be noted that for the above procedures the following methodsand materials were employed:

(i) The G1α (CASHα) deletion mutants and the p55-R/FAS-R (CD120a/CD95)chimera were produced by PCR and/or conventional cloning techniques. TheG1 (CASH) splice variants, the FAS-R (CD95) or p55-R (CD120a)signaling-cascade proteins (all of human origin) and the baculovirus p35protein were expressed in mammalian cells using the pcDNA3 expressionvector (Invitrogen). β-galactosidase was expressed using the pCMV-β-galvector (Promega).

(ii) The human embryonic kidney 293-T and 293 EBNA cells and humancervical carcinoma HeLa cells (HeLa-Fas; the HtTA-1 clone) stablyexpressing transfected human FAS-R (CD95) (established in presentinventor's laboratory) were grown in Dulbecco's modified Eagle's minimalessential medium supplemented with 10% fetal calf serum, nonessentialamino acids, 100 U/ml penicillin and 100 mg/ml streptomycin.

The above findings indicate that G1 can interact with components of thesignaling complexes of p55-R and FAS-R and that it affects deathinduction in a way that may differ depending on the identity of thesplice variant of G1 and on the cell type in which it is expressed.

The inhibition of cytotoxicity induction by G1β, and in the case of theHeLa cells also by G1α, is apparently mediated by the ‘death domain’(DED) region in this protein. It probably reflects competition of theDED of G1 with the corresponding regions in MACH (CASP-8) and Mch4(CASP-10) for binding to MORT1/FADD.

With respect to the way in which CASHα causes death of the 293 cells,the ability of the p35 protein to block this cytotoxic effect indicatesthat the cytotoxicity is mediated by the activity of caspases. However,G1α, even though displaying marked sequence homology to the caspases,may actually lack cysteine-protease activity since it does not haveseveral of the conserved caspase active-site residues. G1α may thereforeact by activating other molecules that do have caspase activity.

Another possibility is that G1α, though unable to act alone as aprotease, can still constitute part of an active protease molecule.Crystallographic studies of CASP-1 and CASP-3 structure indicate thatthe small and large protease subunits in each processed enzyme arederived from distinct proenzyme molecules (Walker et al., 1994; Wilsonet al., 1994; Mittl et al., 1997; Rotonda et al., 1996). In view of theobserved dependence of the G1α cytotoxic activity on intactness of theregion corresponding to the small protease subunit (FIG. 6C), it may bethat this region in G1α (CASHα) can associate with the large subunitregion of certain caspase(s) in a way that results in reconstitution ofan enzymatically active molecule. The resulting active heterotetramershould then be capable of activating other caspases, thus triggeringcell death.

Further, it also arises (results not shown) that G1 has at least somehomology to another protein called MYD88 which is involved in thesignaling pathway mediated by IL-1. Thus, G1 may also be involved inother pathways initiated/mediated by other cytokines.

In view of the above mentioned concerning the cloning and isolation ofG1 protein (at least two isoforms thereof) the following characteristicsand uses of G1 arise:

(i) G1 was cloned by a two-hybrid screen as a molecule that binds theMORT-1-binding protein Mch4 and hence is possibly involved in modulationof the activity of Mch4 and MORT-1, and hence, by the mechanismsindicated above, G1 is possibly involved in the modulation of cellularevents initiated by the FAS-R and p55-R. As Mch4 is capable of bindingto MORT-1 and is directly involved in cell cytotoxicity and ultimatelycell death, and also as some isoforms of Mch4 are known to inhibit celldeath, it arises that G1 including its various isoforms may be directlyor indirectly involved in both cell cytotoxicity and cell death, as wellas, inhibition of cell death.

(ii) G1 apparently has an N-terminal region which contains two MORTmodules homologous to the two MORT modules of MACH (see above referenceExamples 1-3) and of Mch4. The presence of these MORT modules in G1appear therefore to account for G1's ability to bind Mch4, and possiblyalso allow for the binding of G1 (or at least some of its isoforms) toMACH (or some isoforms thereof), as well as, to MORT-1 directly.Accordingly, G1 may be able to modulate the activity of MORT-1 (and, inturn, the activity of FAS-R and p55-R) directly, by direct binding toMORT-1, or indirectly by binding to Mch4 and/or to MACH, which, in turn,are known to bind MORT-1.

(iii) Analysis of the G1 sequence in the above noted databanks andscreening has revealed also that G1 is apparently located close to theMch4 and MACH loci on human chromosome no. 2, indicative of a closerelationship between the genes encoding all of these proteins also atthe chromosomal level.

(iv) At least some of the G1 isoforms (e.g. the G1α isoform of FIG. 1)have a region downstream of the MORT modules region that displayssimilarity with the enzymatic, i.e. protease, region of MACH and Mch4,and as such G1 may be a member of the CED3/ICE protease family.

(v) The presence of a protease-like region in at least some of the G1isoforms (e.g. G1α) indicates that G1 or such isoforms thereof may bedirectly involved in cell cytotoxicity and inflammation caused byvarious stimuli including receptors of the TNF/NGF receptor family (e.g.FAS-R and p55-R) and others as well which act directly or indirectly viaan intracellular protease activity to bring about cell cytotoxicity andinflammation.

(vi) G1 may act as an enhancer or augmentor of the activity of otherproteins, such as, for example, MACH and Mch4 proteins (inclusive oftheir various isoforms), in the intracellular mechanisms leading to cellcytotoxicity, inflammation and other related effects as mediated byreceptors of the TNF/NGF receptor family and others sharing commonintracellular effectors. The enhancer or augmentor effect of G1 (or at+least some of its isoforms) may be by the binding of G1 to these otherproteins (as noted above G1 binds to Mch4 and possibly also binds toMACH and MORT-1), thereby recruiting them to bind to MORT-1 (includingMORT-1 self-association), or to act independently of MORT-1.

(vii) G1 may also act as an inhibitor of the activity of other proteins,and this may be by way of G1 being part of a complex of other proteinsto which it binds (e.g. Mch4 and possibly also MACH and MORT-1) therebyaffecting their cytotoxicity to the extent of inhibition of thisactivity. Further, in an analogous fashion to that mentioned aboveconcerning some MACH isoforms as well as some isoforms of Mch4, theremay also be isoforms of G1 which specifically have inhibitory activity.One such G1 isoform may be the G1β isoform shown in FIG. 2 which has twoMORT MODULES but no apparent protease-like region of similarity to otherknown proteases.

Having now fully described this invention, it will be appreciated bythose skilled in the art that the same can be performed within a widerange of equivalent parameters, concentrations, and conditions withoutdeparting from the spirit and scope of the invention and without undueexperimentation.

While this invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications. This application is intended to cover any variations,uses, or adaptations of the inventions following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth as follows in the scope of theappended claims.

All references cited herein, including journal articles or abstracts,published or corresponding U.S. or foreign patent applications, issuedU.S. or foreign patents, or any other references, are entirelyincorporated by reference herein, including all data, tables, figures,and text presented in the cited references. Additionally, the entirecontents of the references cited within the references cited herein arealso entirely incorporated by reference.

Reference to known method steps, conventional methods steps, knownmethods or conventional methods is not in any way an admission that anyaspect, description or embodiment of the present invention is disclosed,taught or suggested in the relevant art.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingknowledge within the skill of the art (including the contents of thereferences cited herein), readily modify and/or adapt for variousapplications such specific embodiments, without undue experimentation,without departing from the general concept of the present invention.Therefore, such adaptations and modifications are intended to be withinthe meaning and range of equivalents of the disclosed embodiments, basedon the teaching and guidance presented herein. It is to be understoodthat the phraseology or terminology herein is for the purpose ofdescription and not of limitation, such that the terminology orphraseology of the present specification is to be interpreted by theskilled artisan in light of the teachings and guidance presented herein,in combination with the knowledge of one of ordinary skill in the art.

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1. A molecule comprising a DNA sequence encoding a polypeptide which iscapable of binding to one or more of MORT-1, MACH and MORT-1 bindingprotein Mch4, which polypeptide has the amino acid sequence of: (a) a G1protein isoform whose sequence is that of SEQ ID NO:4; (b) a fragment of(a) which is capable of binding to one or more of MORT-1, MACH, andMORT-1 binding protein Mch4; (c) an analog of (a) which differs from thesequence of (a) by no more than ten changes in the amino acid sequenceof (a), each said change being a substitution, deletion and/or insertionof a single amino acid, which analog is capable of binding to one ormore of MORT-1, MACH and MORT-1 binding protein Mch4; or (d) aderivative of (a), (b) or (c) by modification of a functional groupwhich occurs as a side chain or an N- or C-terminal group of one or moreamino acid residues thereof without changing one amino acid to anotherof the twenty commonly occurring natural amino acids, which derivativeis capable of binding to one or more of MORT-1, MACH, and MORT-1 bindingprotein Mch4.
 2. A molecule in accordance with claim 1, wherein saidpolypeptide has the amino acid sequence of said G1 protein isoform ofSEQ ID NO:4
 3. A molecule in accordance with claim 2, wherein the DNAsequence encoding said G1 protein isoform is SEQ ID NO:3.
 4. A moleculein accordance with claim 1, wherein the polypeptide has the amino acidsequence of (c), which is an analog that differs from the sequence of(a) by no more than ten changes in the amino acid sequence of (a), eachsaid change being a deletion and/or insertion of a single amino acid,which analog is capable of binding to one or more of MORT-1, MACH, andMORT-1 binding protein Mch4.
 5. A molecule in accordance with claim 1,wherein the polypeptide has the amino acid sequence of (c), which is ananalog that differs from the sequence of (a) by the substitution,deletion or insertion of a single amino acid residue, which analog iscapable of binding to one or more of MORT-1, MACH, and MORT-1 bindingprotein Mch4.
 6. A molecule in accordance with claim 1, comprising a DNAsequence encoding a G1 isoform whose sequence is that of SEQ ID NO:4,which polypeptide is capable of binding to one or more of MORT-1, MACH,and MORT-1 binding protein Mch4.
 7. A molecule in accordance with claim6, wherein the DNA sequence encoding said G1 protein isoform is that ofSEQ ID NO:3.
 8. A vector comprising the molecule in accordance withclaim
 6. 9. Transformed host cells containing the vector in accordancewith claim
 8. 10. A method for producing a polypeptide which has theamino acid sequence of a G1 protein isoform whose sequence is that ofSEQ ID NO:4, which polypeptide is capable of binding to one or more ofMORT-1, MACH, and MORT-1 binding protein Mch4, comprising: growingtransformed host cells in accordance with claim 9 under conditionssuitable for the expression of an expression product; effectingpost-translational modifications of said expression product as necessaryfor obtaining said polypeptide; and isolating said polypeptide.
 11. Avector comprising the molecule in accordance with claim
 1. 12. A vectorin accordance with claim 11 capable of being expressed in a eukaryotichost cell.
 13. A vector in accordance with claim 11 capable of beingexpressed in a prokaryotic host cell.
 14. Transformed host cellscontaining the vector in accordance with claim
 11. 15. A method forproducing a polypeptide which is capable of binding to one or more ofMORT-1, MACH and MORT-1 binding protein Mch4, comprising: growingtransformed host cells in accordance with claim 14 under conditionssuitable for the expression of an expression product; effectingpost-translational modifications of said expression product as necessaryfor obtaining said polypeptide; and isolating said polypeptide.
 16. Apolypeptide which is capable of binding to one or more of MORT-1, MACHand MORT-1 binding protein Mch4, which polypeptide has the amino acidsequence of: (a) a G1 protein isoform whose sequence is that of SEQ IDNO: 4; (b) a fragment of (a) which is capable of binding to one or moreof MORT-1, MACH, and MORT-1 binding protein Mch4; (c) an analog of (a)which differs from the sequence of (a) by no more than ten changes inthe amino acid sequence of (a), each said change being a substitution,deletion and/or insertion of a single amino acid, which analog iscapable of binding to one or more of MORT-1, MACH, and MORT-1 bindingprotein Mch4; or (d) a derivative of (a), (b) or (c) by modification ofa functional group which occurs as a side chain or an N- or C-terminalgroup of one or more amino acid residues thereof without changing oneamino acid to another of the twenty commonly occurring natural aminoacids, which derivative is capable of binding to one or more of MORT-1,MACH, and MORT-1 binding protein Mch4.
 17. A polypeptide in accordancewith claim 16, having the amino acid sequence of the G1 protein isoformof SEQ ID NO:4.
 18. A polypeptide in accordance with claim 16, havingthe amino acid sequence of an analog which differs from the sequence of(a) by the substitution, deletion or insertion of a single amino acidresidue, which analog is capable of binding to one or more of MORT-1,MACH, and MORT-1 binding protein Mch4.
 19. A polypeptide in accordancewith claim 16, which has the amino acid sequence of a G1 protein isoformwhose sequence is that of SEQ ID NO: 4, which polypeptide is capable ofbinding to one or more of MORT-1 and MACH.
 20. A polypeptide inaccordance with claim 16, having the amino acid sequence of an analogwhich differs from the sequence of (a) by no more than ten changes inthe amino acid sequence of (a), each said change being a deletion and/orinsertion of a single amino acid, which analog is capable of binding toone or more of MORT-1, MACH, and MORT-1 binding protein Mch4.
 21. Apharmaceutical composition for the modulation of the FAS-R ligand- orTNF-effect on cells comprising, as active ingredient, the polypeptideaccording to claim
 16. 22. A molecule comprising a DNA sequence encodinga polypeptide which is capable of binding to one or more of MORT-1, MACHand MORT-1 binding protein Mch4, which polypeptide has the amino acidsequence of: (a) a protein comprising the amino acid sequence ofresidues 1 to 142 of SEQ ID NO:4; (b) a fragment of (a) which is capableof binding to one or more of MORT-1, MACH, and MORT-1 binding proteinMch4; (c) an analog of (a) which differs from the sequence of (a) by nomore than ten changes in the amino acid sequence of (a), each saidchange being a substitution, deletion and/or insertion of a single aminoacid, which analog is capable of binding to one or more of MORT-1, MACHand MORT-1 binding protein Mch4; or (d) a derivative of (a), (b) or (c)by modification of a functional group which occurs as a side chain or anN- or C-terminal group of one or more amino acid residues thereofwithout changing one amino acid to another of the twenty commonlyoccurring natural amino acids, which derivative is capable of binding toone or more of MORT-1, MACH, and MORT-1 binding protein Mch4.
 23. Apolypeptide which is capable of binding to one or more of MORT-1, MACHand MORT-1 binding protein Mch4, which polypeptide has the amino acidsequence of: (a) a protein comprising the amino acid sequence ofresidues 1 to 142 of SEQ ID NO:4; (b) a fragment of (a) which is capableof binding to one or more of MORT-1, MACH, and MORT-1 binding proteinMch4; (c) an analog of (a) which differs from the sequence of (a) by nomore than ten changes in the amino acid sequence of (a), each saidchange being a substitution, deletion and/or insertion of a single aminoacid, which analog is capable of binding to one or more of MORT-1, MACH,and MORT-1 binding protein Mch4; or (d) a derivative of (a), (b) or (c)by modification of a functional group which occurs as a side chain or anN- or C-terminal group of one or more amino acid residues thereofwithout changing one amino acid to another of the twenty commonlyoccurring natural amino acids, which derivative is capable of binding toone or more of MORT-1, MACH, and MORT-1 binding protein Mch4.